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AJP Endocrinology and Metabolism

Impact factor: 4.514 5-Year impact factor: 4.828 Print ISSN: 0193-1849 Online ISSN: 1522-1555 Publisher: American Physiological Society

Subject: Psychology

Most recent papers:

  • Epidermal Growth Factor Receptor Restoration Rescues the Fatty Liver Regeneration in Mice.
    Zimmers, T. A., Jin, X., Zhang, Z., Jiang, Y., Koniaris, L. G.
    AJP Endocrinology and Metabolism. July 11, 2017

    Background: Hepatic steatosis is a common histological finding in obese patients. Even mild steatosis is associated with delayed hepatic regeneration and poor outcomes following liver resection or transplantation. We sought to identify and target molecular pathways that mediate this dysfunction. Material and methods: Lean mice and mice made obese through feeding of a high fat, hyper-caloric diet underwent 70% or 80% hepatectomy. Results: After 70% resection, obese mice demonstrated 100% survival but experienced increased liver injury, reduced energy stores, reduced mitoses, increased necroapoptosis and delayed recovery of liver mass. Increasing liver resection to 80% was associated with mortality of 40% in lean and 80% in obese mice (p<0.05). Gene expression profiling showed decreased Epidermal Growth Factor Receptor (EGFR) in fatty liver. Meta-analysis of expression studies in mice, rats and patients also demonstrated reduction of EGFR in fatty liver. In mice, both EGFR and pEGFR decreased with increasing percentage body fat. Hydrodynamic transfection of EGFR plasmids in mice corrected fatty liver regeneration, reducing liver injury, increasing proliferation and improving survival after 80% resection. Conclusions: loss of EGFR expression is rate limiting for liver regeneration in obesity. Therapies directed at increasing EGFR in steatosis might promote liver regeneration and survival following hepatic resection or transplantation.

    July 11, 2017   doi: 10.1152/ajpendo.00032.2017   open full text
  • Glycemic control after metabolic surgery: a Granger causality and graph analysis.
    Previti, E., Salinari, S., Bertuzzi, A., Capristo, E., Bornstein, S., Mingrone, G.
    AJP Endocrinology and Metabolism. July 11, 2017

    To examine the contribution of Non-Esterified Fatty-Acids (NEFA) and incretin to insulin-resistance and diabetes amelioration after malabsorptive metabolic-surgery that dramatically reduces circulating NEFA. In fact, NEFA infusion reduces glucose-stimulated insulin secretion and high-fat diets predict diabetes development. Six healthy-controls, 11 obese and 10 Type-2-Diabetic (T2D) subjects were studied before and 1 month after Bilio-Pancreatic Diversion (BPD). Twenty-four hours plasma glucose, NEFA, insulin, C-peptide, glucagon-like peptide-1 (GLP1) and gastric-inhibitory-polypeptide (GIP) time courses were obtained and analyzed by Granger causality and graph analyses. Insulin sensitivity and secretion were computed by the oral glucose minimal-model. Before metabolic-surgery NEFA had the strongest influence on the other variables in both obese and T2D subjects. After surgery, GLP1 and C-peptide controlled the system in obese and T2D subjects. Twenty-four hours GIP levels were markedly reduced after BPD. Finally, GLP1 played a central role, but also insulin and C-peptide had a comparable relevance in the network of healthy controls. After BPD insulin sensitivity was completely normalized in both obese and T2D individuals. Increased 24-hours GLP1 circulating levels positively influence glucose homeostasis in both obese and T2D subjects who underwent a malabsorptive bariatric operation. In these latter, the reduction of plasma GIP also contributed to the improvement of glucose metabolism. It is possible that the combination of a pharmaceutical treatment reducing GIP and increasing GLP1 plasma levels will contribute to a better glycemic control in T2D. The application of Granger causality and graph-analyses shed new light on the patho-physiology of metabolic surgery.

    July 11, 2017   doi: 10.1152/ajpendo.00042.2017   open full text
  • MiR-27b augments bone marrow progenitor cell survival via suppressing the mitochondrial apoptotic pathway in type 2 diabetes.
    Li, H., Liu, J., Wang, Y., Fu, Z., Huttemann, M., Monks, T. J., Chen, A. F., Wang, J.-M.
    AJP Endocrinology and Metabolism. July 11, 2017

    Introduction: Bone marrow derived progenitor cells (BMPCs) are potential candidates for autologous cell therapy in tissue repair because of their high angiogenic potential. However, increased progenitor cell apoptosis in diabetes directly limits their success in the clinic. MicroRNAs are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level, but their roles in BMPC-mediated angiogenesis are incompletely understood. In the present study, we tested the hypothesis that the pro-angiogenic miR-27b inhibits BMPC apoptosis in type 2 diabetes. Methods and Results: Bone marrow-derived EPCs from adult male type 2 diabetic db/db mice and control db/+ mice were used. MiR-27b expression (real-time PCR) in EPCs was decreased after 24 hours of exposure to methylglyoxal (MGO) or oxidized Low-Density Lipoprotein. The increase in BMPC apoptosis in the diabetic mice or under MGO exposure was rescued by miR-27b mimic. p53 and the Bax/Bcl-2 ratio in EPCs (Western blot analyses) were significantly higher in diabetic BMPCs, both of which were suppressed by miR-27b. Furthermore, mitochondrial respiration as measured by oxygen consumption rate was enhanced by miR-27b in diabetic BMPCs, with concomitant decrease of mitochondrial Bax/Bcl-2 ratio. The 3'UTR binding assays revealed that both Bax and its activator RUNX1 were direct targets of miR-27b, suggesting that miR-27b inhibits Bax expression in both direct and indirect manners. Conclusion: miR-27b prevents EPC apoptosis in type 2 diabetic mice, at least in part, by suppressing p53 and the Bax/Bcl-2 ratio. These findings may provide a mechanistic basis for rescuing BMPC dysfunction in diabetes for successful autologous cell therapy.

    July 11, 2017   doi: 10.1152/ajpendo.00073.2017   open full text
  • Circulating Sex Steroids Co-regulate Adipose Tissue Immune Cell Populations in Healthy Men.
    Rubinow, K. B., Chao, J., Hagman, D., Kratz, M., Van Yserloo, B., Gaikwad, N. W., Amory, J. K., Page, S. T.
    AJP Endocrinology and Metabolism. July 11, 2017

    Male hypogonadism results in changes in body composition characterized by increases in fat mass. Resident immune cells influence energy metabolism in adipose tissue and could promote increased adiposity through paracrine effects. We hypothesized that manipulation of circulating sex steroid levels in healthy men would alter adipose tissue immune cell populations. Subjects (n=44 men, 19-55 years of age) received 4 weeks of treatment with the GnRH receptor antagonist acyline with daily administration of 1) placebo gel, 2) 1.25g testosterone gel (1.62%), 3) 5g testosterone gel, or 4) 5g testosterone gel with an aromatase inhibitor. Subcutaneous adipose tissue biopsies were performed at baseline and end-of-treatment, and adipose tissue immune cells, gene expression, and intra-adipose estrogen levels were quantified. Change in serum total testosterone level correlated inversely with change in the number of CD3+ (β=-0.36, p=0.04), CD4+ (β=-0.34, p=0.04), and CD8+ (β=-0.33, p=0.05) T cells within adipose tissue. Change in serum 17β-estradiol level correlated inversely with change in the number of adipose tissue macrophages (ATMs) (β=-0.34, p=0.05). A negative association also was found between change in serum testosterone and change in CD11c+ ATMs (β=-0.41, p=0.01). Overall, sex steroid deprivation was associated with increases in adipose tissue T cells and ATMs. No associations were found between changes in serum sex steroid levels and changes in adipose tissue gene expression. Circulating sex steroid levels may regulate adipose tissue immune cell populations. These exploratory findings highlight a possible novel mechanism that could contribute to increased metabolic risk in hypogonadal men.

    July 11, 2017   doi: 10.1152/ajpendo.00075.2017   open full text
  • Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high fat feeding regardless of insulin sensitizing treatment.
    Newsom, S. A., Miller, B. F., Hamilton, K. L., Ehrlicher, S. E., Stierwalt, H. D., Robinson, M. M.
    AJP Endocrinology and Metabolism. July 11, 2017

    Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet, the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 weeks of high-fat compared with low-fat diet. For 8 additional weeks, diets were enriched in pioglitazone to restore insulin sensitivity as compared with non-enriched control low-fat or high-fat diet. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10 to 12. High-resolution respirometry was performed using palmitoyl-L-carnitine, glutamate+malate and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl-L-carnitine oxidation were increased in mice consuming high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H2O2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance.

    July 11, 2017   doi: 10.1152/ajpendo.00144.2017   open full text
  • Maternal methyl donor and cofactor supplementation in late pregnancy increases {beta}-cell numbers at 16 days of life in growth-restricted twin lambs.
    Sulaiman, S. A., De Blasio, M. J., Harland, M. L., Gatford, K. L., Owens, J. A.
    AJP Endocrinology and Metabolism. July 05, 2017

    Restricted growth before birth (IUGR) increases adult risk of Type 2 diabetes by impairing insulin sensitivity and secretion. Altered fetal one-carbon metabolism is implicated in developmental programming of adult health and disease by IUGR. We therefore evaluated effects of maternal dietary supplementation with methyl donors and cofactors (MMDS), designed to increase fetal supply, on insulin action in the spontaneously IUGR twin lamb. In vivo glucose-stimulated insulin secretion and insulin sensitivity were measured at d 12-14 in singleton controls (CON, n=7 lambs from 7 ewes), twins (IUGR, n=8 lambs from 8 ewes), and twins from ewes that received MMDS (2 g rumen-protected methionine, 300 mg folic acid, 1.2 g sulfur, 0.7 mg cobalt) daily from 120 d after mating (~0.8 of term) until delivery (IUGR+MMDS, n=8 lambs from 4 ewes). Body composition and pancreas morphometry were assessed in lambs at d 16. IUGR reduced size at birth and increased neonatal fractional growth rate. MMDS normalized long bone lengths but not other body dimensions of IUGR lambs at birth. IUGR did not impair glucose control or insulin action at d 12-14, compared to controls. MMDS increased metabolic clearance rate of insulin, increased β-cell numerical density and tended to improve insulin sensitivity, compared to untreated IUGR lambs. This demonstrates that effects of late pregnancy methyl donor supplementation persist until at least the third week of life. Whether these effects of MMDS persist beyond early postnatal life and improve metabolic outcomes after IUGR in adults and the underlying mechanisms remain to be determined.

    July 05, 2017   doi: 10.1152/ajpendo.00033.2017   open full text
  • Extra-Ovarian Gonadotropin Negative Feedback Revealed by Aromatase Inhibition in Female Marmoset Monkeys.
    Kraynak, M., Flowers, M. T., Shapiro, R. A., Kapoor, A., Levine, J., Abbott, D. H.
    AJP Endocrinology and Metabolism. July 05, 2017

    While the ovary produces the majority of estradiol (E2) in mature female primates, extra-ovarian sources contribute to E2 synthesis and action, including brain E2 regulating hypothalamic gonadotropin-releasing hormone (GnRH). In ovary-intact female rodent models, aromatase inhibition (AI) induces a PCOS-like hypergonadotropic hyperandrogenism due to absent E2-mediated negative feedback. In order to examine the role of extra-ovarian E2 on nonhuman primate gonadotropin regulation, the present study employs letrozole to elicit AI in adult female marmoset monkeys. Sixteen female marmosets (Callithrix jacchus) (>2yrs) were randomly assigned to ovary intact or ovariectomy (OVX) conditions and subsequently placed on a daily oral regimen of either ~200µl vehicle alone (ovary intact Control, n=3, OVX, n=3) or 1 mg/kg/day Letrozole in vehicle (ovary intact AI, n=4; OVX+AI, n=6). Blood samples were collected every 10 days and plasma chorionic gonadotropin (CG) and steroid hormone levels were determined by validated RIA and LC-MS/MS, respectively. Ovary intact AI-treated and OVX females exhibited elevated CG (p<0.01, p=0.004, respectively) compared with controls, and after 30 days, OVX+AI females exhibited a suprahypergonadotropic phenotype (p=0.004) compared to ovary intact+AI and OVX females. Androstenedione, A4 (p=0.03) and testosterone, T (p=0.05) were also elevated in ovary intact AI-treated females above all other groups. The current study thus confirms in a nonhuman primate that E2 depletion and diminished negative feedback in ovary-intact females engages hypergonadotropic hyperandrogenism. Additionally, we demonstrate that extra-ovarian estrogens, possibly neuroestrogens, contribute to female negative feedback regulation of gonadotropin release.

    July 05, 2017   doi: 10.1152/ajpendo.00058.2017   open full text
  • High, but not low exercise volume, shifts the balance of renin angiotensin system towards ACE2/Mas receptor axis in skeletal muscle in obese rats.
    Frantz, E. D. C., Giori, I. G., Machado, M. V., Magliano, D. C., Freitas, F. M., Andrade, M. S. B., Vieira, A. B., Claudio Lucas da Nobrega, A. C. L., Tibirica, E.
    AJP Endocrinology and Metabolism. July 05, 2017

    Metabolic Syndrome (MS) is a cluster of metabolic risk factors which is linked to central obesity, elevated blood pressure, insulin resistance (IR) and dyslipidemia, where the rennin-angiotensin system (RAS) may provide a link among them. This study aimed to evaluate volume exercise effects comparing low versus high volume of chronic aerobic exercise on RAS axes in skeletal muscle in a diet-induced obesity (DIO) rat model. For this, male Wistar-Kyoto rats were fed a standard chow diet (SC) or a high-fat diet (HF) for 32 weeks. Animals receiving the HF diet were randomly divided into low exercise volume (LEV, 150 min.week-1) and high exercise volume (HEV, 300 min.week-1) at the 20th week. After 12 weeks of aerobic treadmill training the body mass and composition, blood pressure, glucose and lipid metabolism, RAS axes, insulin signaling and inflammatory pathway were performed. HEV slowed the body mass gain, reduced intra-abdominal fat pad and leptin levels, improved total and peripheral body composition and inflammatory cytokine, reduced AT1R expression and increased Mas receptor protein expression compared with the HF animals. Sedentary groups (SC and HF) presented lower time to exhaustion and maximal velocity compared with the LEV and HEV groups. The both exercise training groups showed reduced resting systolic blood pressure and heart rate, improved glucose tolerance, IR, insulin signaling and lipid profile. We conclude that the HEV, but not LEV, shifted the balance of RAS towards the ACE2/Mas receptor axis in skeletal muscle, presenting protective effects against DIO model.

    July 05, 2017   doi: 10.1152/ajpendo.00078.2017   open full text
  • Factors regulating subcutaneous adipose tissue storage, fibrosis, and inflammation may underlie low fatty acid mobilization in insulin sensitive obese adults.
    Van Pelt, D. W., Guth, L. M., Wang, A. Y., Horowitz, J. F.
    AJP Endocrinology and Metabolism. July 05, 2017

    Although the rate of fatty acid release from adipose tissue into the systemic circulation is very high in most obese adults, some obese adults maintain relatively low rates of fatty acid release, which helps protect them against the development of systemic insulin resistance. The primary aim of this study was to identify factors in adipose tissue that may underlie low vs. high rates of fatty acid mobilization in a relatively homogeneous cohort of obese adults. We measured systemic fatty acid rate of appearance (FA Ra) via 13C-palmitate isotope dilution, and we obtained subcutaneous abdominal adipose tissue samples from 30 obese adults (BMI: 38±1 kg/m2, age: 30±2 yr) after an overnight fast. We then measured insulin sensitivity using a hyperinsulinemic-euglycemic clamp. Confirming our previous work, insulin sensitivity was inversely proportional to FA Ra (R2=0.50; p<0.001). Immunoblot analysis of subcutaneous adipose tissue samples revealed that, compared with obese adults with high FA Ra, those with low FA Ra had lower markers of lipase activation and higher abundance of glycerol-3-phosphate acyltransferase (GPAT), which is a primary enzyme for fatty acid esterification. Microarray and pathway analysis provided evidence of lower fibrosis and lower SAPK/JNK pathway activation in obese adults with low FA Ra compared to those with high FA Ra. Our findings suggest that alterations in factors regulating triglyceride storage in adipose tissue, along with lower fibrosis and inflammatory pathway activation, may underlie maintenance of a relatively low FA Ra in obesity, which may help protect against the development of insulin resistance. -

    July 05, 2017   doi: 10.1152/ajpendo.00084.2017   open full text
  • Adaptive Facultative Diet-Induced Thermogenesis in Wild-type but not in UCP1-ablated Mice.
    von Essen, G., Lindsund, E., Cannon, B., Nedergaard, J.
    AJP Endocrinology and Metabolism. July 05, 2017

    The significance of diet-induced thermogenesis (DIT) for metabolic control is still debated. Although obesogenic diets recruit UCP1 and adrenergically inducible thermogenesis, and although the absence of UCP1 may promote the development of obesity, no actual UCP1-related thermogenesis identifiable as diet-induced thermogenesis has to date been unambiguously demonstrated. Examining mice living at thermoneutrality, we have identified a process of facultative (directly elicited by acute eating), adaptive (magnitude develops over weeks on an obesogenic diet) and fully UCP1-dependent thermogenesis. We found no evidence for UCP1-independent diet-induced thermogenesis. The thermogenesis was proportional to the total amount of UCP1 protein in brown adipose tissue and was not dependent on any contribution of UCP1 in brite/beige adipose tissue, since no UCP1 protein was found there under these conditions. Total UCP1 protein amount developed proportionally to total body fat content. The physiological messenger linking obesity level and acute eating to increased thermogenesis is not known. Thus, UCP1-dependent diet-induced thermogenesis limits obesity development during exposure to obesogenic diets but does not prevent obesity as such.

    July 05, 2017   doi: 10.1152/ajpendo.00097.2017   open full text
  • The bradykinin(BK)-cGMP-PKG pathway augments insulin sensitivity via upregulation of MAPK phosphatase-5 and inhibition of Jun kinase (JNK).
    Frigolet, M. E., Thomas, G., Beard, K., Lu, H., Liu, L., Fantus, I. G.
    AJP Endocrinology and Metabolism. July 05, 2017

    Bradykinin (BK) promotes insulin sensitivity and glucose uptake in adipocytes and other cell types. We demonstrated that in rat adipocytes BK enhances insulin-stimulated glucose transport via endothelial nitric oxide synthase (eNOS), nitric oxide (NO) generation, and decreased activity of the mitogen activated protein kinase (MAPK), JNK (c-Jun-N-terminal kinase). In endothelial cells, NO increases soluble guanylate cyclase (sGC) activity which in turn, activates protein kinase G (PKG) by increasing cGMP levels. In this study, we investigated whether BK acts via the sGC-cGMP-PKG pathway to inhibit the negative effects of JNK on insulin signaling and glucose uptake in rat adipocytes. BK augmented cGMP concentrations. The BK-induced enhancement of insulin-stimulated glucose uptake was mimicked by the sGC activator, YC-1 and a cell permeable cGMP analog, CPT-cGMP, and inhibited by the sGC inhibitor, ODQ and the PKG inhibitor, KT-5823. Transfection of dominant-negative PKG reduced the BK augmentation of insulin-induced Akt phosphorylation. The activation of JNK and ERK1/2 by insulin was attenuated by BK, which was mediated by the sGC-cGMP-PKG pathway. While insulin-stimulated phosphorylation of upstream activators of JNK and ERK, i.e., MKK4 and MEK1/2, was unaffected, BK augmented insulin-mediated induction of MKP-5 mRNA and protein levels. Furthermore, zaprinast, a phophodiesterase inhibitor, enhanced cGMP and MKP-5 and prolonged the action of BK. These data indicate that BK enhances insulin action by inhibition of negative feedback by JNK and ERK via upregulation of MKP-5, mediated by the sGC-cGMP-PKG signaling pathway.

    July 05, 2017   doi: 10.1152/ajpendo.00298.2016   open full text
  • Incretin Secretion in Humans is under the Influence of Cannabinoid Receptors.
    Chia, C. W., Carlson, O. D., Liu, D. D., Gonzalez-Mariscal, I., Santa-Cruz Calvo, S., Egan, J. M.
    AJP Endocrinology and Metabolism. June 27, 2017

    The mechanisms regulating incretin secretion are not fully known. Human obesity is associated with altered incretin secretion and elevated endocannabinoid levels. Since cannabinoid receptors (CBRs) are expressed on incretin-secreting cells in rodents, we hypothesized that endocannabinoids are involved in the regulation of incretin secretion. We compared plasma glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) responses during oral glucose tolerance test (OGTT) in twenty lean and twenty obese participants from the Baltimore Longitudinal Study of Aging (BLSA). Next, we recruited 20 healthy men to evaluate GIP and GLP-1 responses during OGTT after administering placebo or nabilone (CBR agonist), in a randomized, double-blind, crossover fashion. Compared to the BLSA lean group, the BLSA obese group had significantly higher fasting and post-OGTT GIP levels, but similar fasting GLP-1 and significantly lower post-OGTT GLP-1 levels. In the nabilone versus placebo study, when compared to placebo, nabilone resulted in significantly elevated post-dose fasting GIP levels and post-OGTT GIP levels, but no change in post-dose fasting GLP-1 levels together with a significantly lower post-OGTT GLP-1 levels. Glucose levels were not different with both interventions. We conclude that elevated GIP levels in obesity are likely a consequence of increased endocannabinoid levels. CBRs exert tonic control over GIP secretion, which may have a homeostatic effect in suppressing GLP-1 secretion. This raises the possibility that gut hormones are influenced by endocannabinoids.

    June 27, 2017   doi: 10.1152/ajpendo.00080.2017   open full text
  • Increased adipose tissue aromatase activity improves insulin sensitivity and reduces adipose tissue inflammation in male mice.
    Ohlsson, C., Hammarstedt, A., Vandenput, L., Saarinen, N., Ryberg, H., Windahl, S., Farman, H. H., Janson, J.-O., Moverare-Skrtic, S., Smith, U., Zhang, F.-P., Poutanen, M., Hedjazifar, S., Sjogren, K.
    AJP Endocrinology and Metabolism. June 27, 2017

    Females are in general more insulin sensitive than males. To investigate if this is a direct effect of sex-steroids (SS) in white adipose tissue (WAT), we developed a male mouse model over expressing the aromatase enzyme, converting testosterone (T) to estradiol (E2), specifically in WAT (Ap2-arom mice). Adipose tissue E2 levels were increased while circulating SS levels were unaffected in male Ap2-arom mice. Importantly, male Ap2-arom mice were more insulin sensitive compared with WT mice and exhibited increased serum adiponectin levels and upregulated expression of Glut4 and Irs1 in WAT. The expression of markers of macrophages and immune cell infiltration was markedly decreased in WAT of male Ap2-arom mice. The adipogenesis was enhanced in male Ap2-arom mice, supported by elevated Pparg expression in WAT and enhanced differentiation of pre-adipocyte into mature adipocytes. In summary, increased adipose tissue aromatase activity reduces adipose tissue inflammation and improves insulin sensitivity in male mice. We propose that estrogen increases insulin sensitivity via a local effect in WAT on adiponectin expression, adipose tissue inflammation, and adipogenesis.

    June 27, 2017   doi: 10.1152/ajpendo.00093.2017   open full text
  • Deletion of UCP1 enhances ex vivo aortic vasomotor function in female but not male mice despite similar susceptibility to metabolic dysfunction.
    Winn, N. C., Grunewald, Z. I., Gastecki, M. L., Woodford, M. L., Welly, R. J., Clookey, S. L., Ball, J. R., Gaines, T. L., Karasseva, N. G., Kanaley, J. A., Sacks, H. S., Vieira-Potter, V. J., Padilla, J.
    AJP Endocrinology and Metabolism. June 27, 2017

    Females are typically more insulin sensitive than males which may be partly attributed to greater brown adipose tissue (BAT) activity and uncoupling protein 1 (UCP1) content. Accordingly, we tested the hypothesis that UCP1 deletion would abolish sex differences in insulin sensitivity and that whitening of thoracic periaortic BAT caused by UCP1 loss would be accompanied with impaired thoracic aortic function. Furthermore, because UCP1 exerts antioxidant effects, we examined whether UCP1 deficiency-induced metabolic dysfunction was mediated by oxidative stress. Compared to males, female mice had lower HOMA- and AT-insulin resistance (IR) despite no significant differences in BAT UCP1 content. UCP1 ablation increased HOMA-IR, AT-IR, and whitening of BAT in both sexes. Expression of UCP1 in thoracic aorta was greater in wild-type females compared to males. Importantly, deletion of UCP1 enhanced aortic vasomotor function in females only. UCP1 ablation did not promote oxidative stress in interscapular BAT. Furthermore, daily administration of the free radical scavenger tempol for 8 weeks did not abrogate UCP1 deficiency-induced increases in adiposity, hyperinsulinemia or liver steatosis. Collectively, we report that: 1) in normal chow-fed mice housed at 25°C, aortic UCP1 content was greater in females than males and its deletion improved ex vivo aortic vasomotor function in females only; 2) constitutive UCP1 content in BAT was similar between females and males and loss of UCP1 did not abolish sex-differences in insulin sensitivity; and 3) the metabolic disruptions caused by UCP1 ablation did not appear to be contingent upon increased oxidative stress in mice under normal dietary conditions.

    June 27, 2017   doi: 10.1152/ajpendo.00096.2017   open full text
  • Lipid remodelling and an altered membrane proteome may drive the effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion.
    Jeromson, S., Mackenzie, I., Doherty, M. K., Whitfield, P. D., Bell, G., Dick, J., Shaw, A., Rao, F., Ashcroft, S., Philp, A., Galloway, S., Gallagher, I., Hamilton, D. L.
    AJP Endocrinology and Metabolism. June 27, 2017

    In striated muscle, EPA and DHA have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C2C12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment on the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. FAME analysis revealed that both EPA and DHA led to similar but substantial changes in fatty acid profiles. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long chain fatty acids with 5 (EPA treatment) or 6 (DHA treatment) double bonds. As these are typically membrane associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. SILAC based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane associated proteome. We conclude that the EPA specific increase in polyunsaturated long chain fatty acids in the phospholipid fraction is associated with an altered membrane associated proteome and these may be critical events in the metabolic remodelling induced by EPA treatment.

    June 27, 2017   doi: 10.1152/ajpendo.00438.2015   open full text
  • Interrelations between 3-hydroxypropionate and propionate metabolism in rat liver: Relevance to disorders of propionyl-CoA metabolism.
    Wilson, K. A., Han, Y., Zhang, M., Hess, J., Chapman, K. A., Cline, G. W., Tochtrop, G. P., Brunengraber, H., Zhang, G.-F.
    AJP Endocrinology and Metabolism. June 20, 2017

    Propionate, 3-hydroxypropionate (3HP), methylcitrate, related compounds and ammonium accumulate in body fluids of patients with disorders of propionyl-CoA metabolism, such as propionic acidemia. Although liver transplantation alleviates hyperammonemia, high concentrations of propionate, 3HP and methylcitrate persist in body fluids. We hypothesized that conserved metabolic perturbations occurring in transplanted patients result from the simultaneous presence of propionate and 3HP in body fluids. We investigated the interrelations of propionate and 3HP metabolism in perfused livers from normal rats using metabolomic and stable isotopic technologies. In the presence of propionate, 3HP or both, we observed the following metabolic perturbations. First, the citric acid cycle (CAC) is overloaded, but does not provide sufficient reducing equivalents to the respiratory chain to maintain the homeostasis of adenine nucleotides. Second, there is major CoA trapping in the propionyl-CoA pathway, and a tripling of liver total CoA within 1 hr. Third, liver proteolysis is stimulated. Fourth, propionate inhibits the conversion of 3HP to acetyl-CoA and its oxidation in the CAC. Fifth, some propionate and some 3HP are converted to nephrotoxic maleate by different processes. Our data have implication for the clinical management of propionic acidemia. They also emphasize the perturbations of liver intermediary metabolism induced by supraphysiological i.e., mM concentrations of labeled propionate used to trace intermediary metabolism, in particular inhibition of CAC flux and major decreases in the [ATP]/[ADP] and [ATP]/[AMP] ratios.

    June 20, 2017   doi: 10.1152/ajpendo.00105.2017   open full text
  • MAP4K4 impairs energy metabolism in endothelial cells and promotes insulin resistance in obesity.
    Roth Flach, R. J., DiStefano, M. T., Danai, L. V., Senol-Cosar, O., Yawe, J. C., Kelly, M., Garcia Menendez, L., Czech, M. P.
    AJP Endocrinology and Metabolism. June 13, 2017

    The blood vasculature responds to insulin, influencing hemodynamic changes in the periphery, which promotes tissue nutrient and oxygen delivery and thus metabolic function. The lymphatic vasculature regulates fluid and lipid homeostasis, and impaired lymphatic function can contribute to atherosclerosis and obesity. Recent studies have suggested a role for endothelial cell (EC) Mitogen activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and lymphangiogenesis as well as atherosclerosis. Here, we show that inducible EC Map4k4 deletion in adult mice ameliorates metabolic dysfunction in obesity despite the development of chylous ascites and a concomitant striking increase in adipose tissue lymphocyte content. Despite these defects, animals lacking endothelial Map4k4 were protected from skeletal muscle microvascular rarefaction in obesity, and primary ECs lacking Map4k4 displayed reduced senescence and increased metabolic capacity. Thus, endothelial Map4k4 has complex and opposing functions in the blood and lymphatic endothelium post-development. Whereas blood endothelial Map4k4 promotes vascular dysfunction and impairs glucose homeostasis in adult animals, lymphatic endothelial Map4k4 is required to maintain lymphatic vascular integrity and regulate immune cell trafficking in obesity.

    June 13, 2017   doi: 10.1152/ajpendo.00037.2017   open full text
  • The Intestinal Renal Axis For Arginine Synthesis Is Present And Functional In The Neonatal Pig.
    Marini, J. C., Agarwal, U., Robinson, J. L., Yuan, Y., Cajo Didelija, I., Stoll, B., Burrin, D. G.
    AJP Endocrinology and Metabolism. June 13, 2017

    The intestinal-renal axis for endogenous arginine synthesis is an interorgan process in which citrulline produced in the small intestine is utilized by the kidney for arginine synthesis. The function of this axis in neonates has been questioned because during this period the enzymes needed for arginine synthesis argininosuccinate synthase and lyase (ASS1 and ASL) are present in the gut. However, evidence of high plasma citrulline concentrations in neonates suggests otherwise. We quantified in vivo citrulline production in premature (10 d preterm), neonatal (7 d old) and young pigs (35 d old) using citrulline tracers. Neonatal pigs had higher fluxes (69 µmol•kg-1•h-1; P <0.001) than premature and young pigs (43 and 45 µmol•kg-1•h-1, respectively). Plasma citrulline concentration was also greater in neonatal pigs than in the other age groups. We also determined the site of synthesis and utilization of citrulline in neonatal and young pigs, measuring organ balances across the gut and the kidney. Citrulline was released from the gut and utilized by the kidney in both neonatal and young pigs. The abundance and localization of the enzymes involved in the synthesis and utilization were determined in intestinal and kidney tissue. Despite the presence of ASS1 and ASL in the neonatal small intestine, the lack of co-localization with the enzymes that produce citrulline results in the release of citrulline by the portal drained viscera and its utilization by the kidney to produce arginine. In conclusion, the intestinal-renal axis for arginine synthesis is present in the neonatal pig.

    June 13, 2017   doi: 10.1152/ajpendo.00055.2017   open full text
  • CNS {beta}3-adrenergic receptor activation regulates feeding behavior, white fat browning and body weight.
    Richard, J. E., Lopez-Ferreras, L., Chanclon, B., Eerola, K., Micallef, P., Skibicka, K. P., Wernstedt Asterholm, I.
    AJP Endocrinology and Metabolism. June 06, 2017

    Pharmacological β3-adrenergic receptor (β3AR) activation leads to increased mitochondrial biogenesis and activity in white adipose tissue (WAT), a process commonly referred to as "browning", and transiently increased insulin release. These effects are associated with improved metabolic function and weight loss. It is assumed that this impact of β3AR agonists is mediated solely through activation of β3ARs in adipose tissue. However, β3ARs are also found in the brain, in areas such as the brainstem and hypothalamus that provide multisynaptic innervation to brown and white adipose depots. Thus, contrary to the current adipocentric view, the CNS may also have the ability to regulate energy balance and metabolism through actions on central β3ARs. This study therefore aimed to elucidate whether CNS β3ARs can regulate browning of WAT and other aspects of metabolic regulation such as food intake control and insulin release. We found that acute central injection of β3AR agonist potently reduced food intake, body weight, and increased hypothalamic neuronal activity in rats. Acute central β3AR stimulation was also accompanied by a transient increase in circulating insulin levels. Moreover, subchronic central β3AR agonist treatment led to a browning response in both inguinal (IWAT) and gonadal WAT (GWAT), along with reduced GWAT and increased BAT mass. In high fat-high sugar fed rats subchronic central β3AR stimulation reduced body weight, chow, lard, and sucrose water intake, in addition to increasing browning of IWAT and GWAT. Collectively, our results identify the brain as a new site of action for the anorexic and browning impact of β3AR-activation.

    June 06, 2017   doi: 10.1152/ajpendo.00418.2016   open full text
  • High-Intensity Interval Training and Calorie Restriction Promote Remodeling of Glucose and Lipid Metabolism in Diet-Induced Obesity.
    Davis, R. A. H., Halbrooks, J. E., Watkins, E., Fisher, G., Hunter, G. R., Nagy, T. R., Plaisance, E. P.
    AJP Endocrinology and Metabolism. June 06, 2017

    Calorie restriction (CR) decreases adiposity, but the magnitude and defense of weight loss is less than predicted due to reductions in total daily energy expenditure (TEE). The purpose of the current investigation was to determine if high-intensity interval training (HIIT) would increase markers of sympathetic activation in white adipose tissue (WAT) and rescue CR-mediated reductions in EE to a greater extent than moderate-intensity aerobic exercise training (MIT). Thirty-two 5-wk old male C57BL/6J mice were placed on ad libitum HFD for 11 weeks followed by randomization to one of four groups (n = 8 per group) for an additional 15 weeks: 1) CON (remain on HFD); 2) CR (25% lower energy intake); 3) CR+HIIT (25% energy deficit created by 12.5% CR and 12.5% EE through HIIT); and 4) CR+MIT (25% energy deficit created by 12.5% CR and 12.5% EE through MIT). Markers of adipose thermogenesis (Ucp1, Prdm16, Dio2, Fgf21) were unchanged in either exercise group in inguinal or epididymal WAT while CR+HIIT decreased Ucp1 expression in retroperitoneal WAT and brown adipose tissue. HIIT rescued CR-mediated reductions in lean body mass (LBM) and resting energy expenditure (REE) and both were associated with improvements in glucose/insulin tolerance. Improvements in glucose metabolism in the CR+HIIT group appear to be linked to a molecular signature which enhances glucose and lipid storage in skeletal muscle. These findings suggest that negative energy balance abolishes exercise-mediated increases in markers of WAT thermogenesis but remodels skeletal muscle metabolic and thermogenic capacity which are linked to enhanced glucose metabolism.

    June 06, 2017   doi: 10.1152/ajpendo.00445.2016   open full text
  • Hepatic FGF21 mediates gender differences in high-fat high-fructose diet induced fatty liver.
    Chukijrungroat, N., Khamphaya, T., Weerachayaphorn, J., Songserm, T., Saengsirisuwan, V.
    AJP Endocrinology and Metabolism. May 30, 2017

    The role of gender in the progression of fatty liver due to chronic high-fat, high-fructose diet (HFFD) has not been studied. The present investigation assessed whether HFFD induced hepatic perturbations differently between genders and examined the potential mechanisms. Male, female and ovariectomized (OVX) Sprague-Dawley rats were fed either control diet or HFFD for 12 weeks. Indices of liver damage and hepatic steatosis were analyzed biochemically and histologically together with monitoring changes in hepatic gene and protein expression. HFFD induced a higher degree of hepatic steatosis in females with significant increases in proteins involved in hepatic lipogenesis. Whereas, HFFD significantly induced liver injury, inflammation, and oxidative stress only in males. Interestingly, a significant increase in hepatic fibroblast growth factor 21 (FGF21) protein expression was observed in HFFD-fed males but not in HFFD-fed females. Ovarian hormone deprivation by itself led to a significant reduction in FGF21 with hepatic steatosis, and HFFD further aggravated hepatic fat accumulation in OVX rats. Importantly, estrogen replacement restored hepatic FGF21 levels and reduced hepatic steatosis in HFFD-fed OVX rats. Collectively, our results indicate that male rats are more susceptible to HFFD-induced hepatic inflammation, and the mechanism underlying this sex dimorphism is mediated through hepatic FGF21 expression. Our findings reveal gender differences in the development of HFFD-induced fatty liver and indicate the protective role of estrogen against HFFD-induced hepatic steatosis.

    May 30, 2017   doi: 10.1152/ajpendo.00076.2017   open full text
  • Fibroblast growth factor 21: a regulator of metabolic disease and health span.
    Xie, T., Leung, P. S.
    AJP Endocrinology and Metabolism. May 30, 2017

    Fibroblast growth factor 21 (FGF21) is a potent endocrine regulator with physiological effects on glucose and lipid metabolism and thus garners much attention on its translational potential for the management of obesity and related metabolic syndromes. FGF21 is mainly expressed in several metabolically active tissue organs, such as the liver, adipose tissue, skeletal muscle and pancreas with profound effects and therapeutically relevance. Emerging experimental and clinical data point to the demonstrated metabolic benefits of FGF21 which includes, but not limit to, weight loss, glucose and lipid metabolism as well as insulin sensitivity. Besides, FGF21 also acts directly through its co-receptor β-klotho in the brain to alter light-dark cycle activity. In this review, we critically appraise the current advance in the physiological actions of FGF21 and its role as a biomarker of various metabolic diseases, especially type 2 diabetes mellitus (T2DM). We also discuss the potential exciting role of FGF21 in improving our health and prolonging our life span. This information will provide a fuller understanding for further researches into FGF21, as well as providing a scientific basis for potential establishing a healthcare guideline of this promising molecule.

    May 30, 2017   doi: 10.1152/ajpendo.00101.2017   open full text
  • Glucagon's effect on liver protein metabolism in vivo.
    Kraft, G., Coate, K. C., Winnick, J. J., Dardevet, D., Donahue, E. P., Cherrington, A. D., Williams, P. E., Moore, M. C.
    AJP Endocrinology and Metabolism. May 23, 2017

    The postprandial state is characterized by a storage of nutrients in the liver, muscle and adipose tissue for later utilization. In the case of a protein rich meal, amino acids (AA) stimulate glucagon secretion by the α-cell. The aim of the present study was to determine the impact of the rise in glucagon on AA metabolism, particularly in the liver. We used a conscious catheterized dog model to recreate a postprandial condition using a pancreatic clamp. Portal infusions of glucose, AA and insulin were used to achieve postprandial levels while portal glucagon infusion either maintained the basal level or increased it by 3 fold. The high glucagon infusion reduced the increase in arterial AA concentrations compared to the basal glucagon level (-23%, P<0.05). In the presence of high glucagon, liver AA metabolism shifted towards a more catabolic state with less protein synthesis (-36%) and increased net urea production (+52%). Net hepatic glucose uptake was reduced (-35%), in association with lower glycogen synthesis (-54%), and also in part because of a higher utilization of AA in gluconeogenesis. The phosphorylation of AMPK was increased by the high glucagon infusion (+40%) and this could be responsible for increasing the expression of genes related to pathways producing energy and lowering those involved in energy consumption. In conclusion, the rise in glucagon associated with a protein rich meal promotes a catabolic utilization of AA in the liver thereby opposing the storage of AA in proteins.

    May 23, 2017   doi: 10.1152/ajpendo.00045.2017   open full text
  • The NLRP3 inflammasome contributes to sarcopenia and lower muscle glycolytic potential in old mice.
    McBride, M. J., Foley, K. P., D'souza, D. M., Li, Y. E., Lau, T. C., Hawke, T. J., Schertzer, J. D.
    AJP Endocrinology and Metabolism. May 23, 2017

    The mechanisms underpinning decreased skeletal muscle strength and slowing of movement during aging are ill-defined. "Inflammaging" - increased inflammation with advancing age - may contribute to aspects of sarcopenia, but little is known about the participatory immune components. We discovered that aging was associated with increased caspase-1 activity in mouse skeletal muscle. We hypothesized that the caspase-1 containing NLRP3 inflammasome contributes to sarcopenia in mice. Male C57BL/6J wild type (WT) and NLRP3-/- mice were aged to 10 months (adult) and 24 months (old). NLRP3-/- mice were protected from decreased muscle mass (relative to body mass) and decreased size of Type IIB and IIA myofibers, which occurred between 10 and 24 months of age in WT mice. Old NLRP3-/- mice also had increased relative muscle strength and endurance and were protected from age-related increases in the number of myopathic fibers. We found no evidence of age-related or NLRP3-dependent changes in markers of systemic inflammation. Increased caspase-1 activity was associated with GAPDH proteolysis and reduced GAPDH enzymatic activity in skeletal muscles from old WT mice. Aging did not alter caspase-1 activity, GAPDH proteolysis or GAPDH activity in skeletal muscles of NLRP3-/- mice. Our results show that the NLRP3 inflammasome participates in age-related loss of muscle glycolytic potential. Deletion of NLRP3 mitigates both the decline in glycolytic myofiber size and the reduced activity of glycolytic enzymes in muscle during aging. We propose that the etiology of sarcopenia involves direct communication between immune responses and metabolic flux in skeletal muscle.

    May 23, 2017   doi: 10.1152/ajpendo.00060.2017   open full text
  • Pre-sleep dietary protein-derived amino acids are incorporated in myofibrillar protein during post-exercise overnight recovery.
    Trommelen, J., Kouw, I. W. K., Holwerda, A. M., Snijders, T., Halson, S. L., Rollo, I., Verdijk, L. B., van Loon, L. J. C.
    AJP Endocrinology and Metabolism. May 23, 2017

    The purpose of this study was to determine the impact of ingesting 30 g casein protein with and without 2 g free leucine prior to sleep on myofibrillar protein synthesis rates during post-exercise overnight recovery. 36 healthy young males performed a single bout of resistance-type exercise in the evening (19:45 h) after a full day of dietary standardization. Thirty min prior to sleep (23:30 h), subjects ingested 30 g intrinsically L-[1-13C]-phenylalanine-labeled protein with (PRO+leu, n=12) or without (PRO, n=12) 2 g free leucine, or a noncaloric placebo (PLA, n=12). Continuous intravenous L-[ring-2H5]-phenylalanine, L-[1-13C]-leucine and L-[ring-2H2]-tyrosine infusions were applied. Blood and muscle tissue samples were collected to assess whole-body protein net balance, myofibrillar protein synthesis rates and overnight incorporation of dietary protein-derived amino acids into myofibrillar protein. Protein ingestion prior to sleep improved overnight whole-body protein net balance (P<0.001). Myofibrillar protein synthesis rates did not differ significantly between treatments as assessed by L-[ring-2H5]-phenylalanine (0.057±0.002, 0.055±0.002, and 0.055±0.004 %•h-1 for PLA, PRO, and PRO+leu, respectively; P=0.850) or L-[1-13C]-leucine (0.080±0.004, 0.073±0.004, and 0.083±0.006 %•h-1, respectively; P=0.328). Myofibrillar L-[1-13C]-phenylalanine enrichments increased following protein ingestion, but did not differ between the PRO and PRO+leu treatments. In conclusion, protein ingestion prior to sleep improves whole-body protein net balance and provides amino acids that are incorporated into myofibrillar protein during sleep. However, the ingestion of 30 g casein protein with or without additional free leucine prior to sleep does not increase muscle protein synthesis rates during post-exercise overnight recovery.

    May 23, 2017   doi: 10.1152/ajpendo.00273.2016   open full text
  • Glucose Autoregulation is the Dominant Component of the Hormone-Independent Counterregulatory Response to Hypoglycemia in the Conscious Dog.
    Gregory, J. M., Rivera, N., Kraft, G., Winnick, J. J., Farmer, B., Allen, E. J., Donahue, E. P., Smith, M. S., Edgerton, D. S., Williams, P. E., Cherrington, A. D.
    AJP Endocrinology and Metabolism. May 16, 2017

    The contribution of hormone-independent counterregulatory signals in defense of insulin-induced hypoglycemia were determined in adrenalectomized, overnight-fasted conscious dogs receiving hepatic portal vein insulin infusions at a rate 20-fold basal. Either euglycemia was maintained (group 1) or hypoglycemia (45 mg/dL) was allowed to occur. There were 3 hypoglycemic groups: one in which hepatic autoregulation against hypoglycemia occurred in the absence of sympathetic nervous system input (group 2), autoregulation in the presence of norepinephrine (NE) signaling to fat and muscle (group 3), and autoregulation in the presence of NE signaling to fat, muscle, and liver (group 4). Average net hepatic glucose balance (NHGB) during the last hour for groups 1-4 was -0.7±0.1, 0.3±0.1 (p<0.01 vs. group1), 0.7±0.1 (p=0.01 vs. group 2), and 0.8±0.1 (p=0.7 vs group 3) mg/kg/min, respectively. Hypoglycemia per se (group 2) increased NHGB by causing an inhibition of net hepatic glycogen synthesis. NE signaling to fat and muscle (group 3) increased NHGB further by mobilizing gluconeogenic precursors resulting in a rise in gluconeogenesis. Lowering glucose per se decreased non-hepatic glucose uptake by 8.9 mg/kg/min and the addition of increased neural efferent signaling to muscle and fat blocked glucose uptake further by 3.2 mg/kg/min. The addition of increased neural efferent input to liver did not affect NHGB or non-hepatic glucose uptake significantly. In conclusion, even in the absence of increases in counterregulatory hormones, the body can defend itself against hypoglycemia using glucose autoregulation and increased neural efferent signaling both of which stimulate hepatic glucose production and limit glucose utilization.

    May 16, 2017   doi: 10.1152/ajpendo.00099.2017   open full text
  • Functional High Intensity Training Improves Pancreatic {beta}-cell Function in Adults with Type 2 Diabetes.
    Nieuwoudt, S., Fealy, C. E., Foucher, J. A., Scelsi, A. R., Malin, S. K., Pagadala, M. R., Rocco, M., Burguera, B., Kirwan, J. P.
    AJP Endocrinology and Metabolism. May 16, 2017

    Type 2 diabetes (T2D) is characterized by reductions in β-cell function and insulin secretion on the background of elevated insulin resistance. Aerobic exercise has been shown to improve β-cell function, despite a subset of T2D patients displaying "exercise resistance". Further investigations into the effectiveness of alternate forms of exercise on β-cell function in the T2D patient population are needed. We examined the effect of a novel 6-week CrossFit™ Functional High Intensity Training (F-HIT) intervention on β-cell function in 12 sedentary adults with clinically diagnosed T2D (54±2 years, 166±16 mg/dL fasting glucose). Supervised training was completed 3 days a week, comprising of functional movements performed at a high intensity in a variety of 10-20 minute sessions. All subjects completed an oral glucose tolerance test and anthropometric measures at baseline and following the intervention. The mean Disposition Index (DI), a validated measure of β-cell function, was significantly increased (PRE: 8.4±3.1, POST: 11.5±3.5, P=0.02) after the intervention. Insulin processing inefficiency in the β-cell, expressed as the fasting proinsulin-to-insulin ratio, was also reduced (PRE: 2.40±0.37, POST: 1.78±0.30, P=0.04). Increased β-cell function during the early-phase response to glucose correlated significantly with reductions in abdominal body fat (R2=0.56, P=0.005) and fasting plasma alkaline phosphatase (R2=0.55, P=0.006). Mean total body fat percentage decreased significantly (: -1.17±0.30%, P=0.003), while lean body mass was preserved (: +0.05±0.68kg, P=0.94). We conclude that F-HIT is an effective exercise strategy for improving β-cell function in adults with T2D.

    May 16, 2017   doi: 10.1152/ajpendo.00407.2016   open full text
  • Effects of metformin on compensatory pancreatic {beta}-cell hyperplasia in mice fed a high-fat diet.
    Tajima, K., Shirakawa, J., Okuyama, T., Kyohara, M., Yamazaki, S., Togashi, Y., Terauchi, Y.
    AJP Endocrinology and Metabolism. May 16, 2017

    Metformin has been widely used for the treatment of type 2 diabetes. However, the effect of metformin on pancreatic β-cells remains controversial. In this study, we investigated the impacts of treatment with metformin on pancreatic β-cells in a mouse model fed a high-fat diet (HFD), which triggers adaptive β-cell replication. An 8-week treatment with metformin improved insulin resistance and suppressed the compensatory β-cell hyperplasia induced by HFD-feeding. In contrast, the increment in β-cell mass arising from 60 weeks of HFD-feeding was similar in mice treated with and those treated without metformin. Interestingly, metformin suppressed β-cell proliferation induced by 1 week of HFD-feeding without any changes in insulin resistance. Metformin directly suppressed glucose-induced β-cell proliferation in islets and INS-1 cells in accordance with a reduction in mTOR signaling. Taken together, metformin suppressed HFD-induced β-cell proliferation independent of the improvement of insulin resistance, partly via direct actions.

    May 16, 2017   doi: 10.1152/ajpendo.00447.2016   open full text
  • The Niemann-Pick C1 gene interacts with a high-fat diet to promote weight gain through differential regulation of central energy metabolism pathways.
    Castillo, J. J., Jelinek, D., Wei, H., Gannon, N. P., Vaughan, R. A., Horwood, L. J., Meaney, F. J., Garcia-Smith, R., Trujillo, K. A., Heidenreich, R. A., Meyre, D., Orlando, R. A., LeBoeuf, R. C., Garver, W. S.
    AJP Endocrinology and Metabolism. May 09, 2017

    A genome-wide association study (GWAS) reported that common variation in the human Niemann-Pick C1 gene (NPC1) is associated with morbid adult obesity. This study was confirmed using our BALB/cJ Npc1 mouse model, whereby heterozygous mice (Npc1+/-) with decreased gene dosage were susceptible to weight gain when fed a high-fat diet (HFD) compared to homozygous normal mice (Npc1+/+) fed the same diet. The objective for our current study was to validate this Npc1 gene-diet interaction using statistical modeling with fitted growth trajectories, conduct body weight analyses for different measures, and define the physiological basis responsible for weight gain. Metabolic phenotype analysis indicated no significant difference between Npc1+/+ and Npc1+/- mice fed a HFD for food and water intake, oxygen consumption, carbon dioxide production, locomotor activity, adaptive thermogenesis, and intestinal lipid absorption. However, the livers from Npc1+/- mice had significantly increased amounts of mature sterol regulatory element-binding protein-1 (SREBP-1) and increased expression of SREBP-1 target genes that regulate glycolysis and lipogenesis with an accumulation of triacylglycerol and cholesterol. Moreover, white adipose tissue from Npc1+/- mice had significantly decreased amounts of phosphorylated hormone sensitive lipase (pHSL) with decreased triacylglycerol lipolysis. Consistent with these results, cellular energy metabolism studies indicated that Npc1+/- fibroblasts had significantly increased glycolysis and lipogenesis in addition to significantly decreased substrate (glucose and endogenous fatty acid) oxidative metabolism with an accumulation of triacylglycerol and cholesterol. In conclusion, these studies demonstrate that the Npc1 gene interacts with a HFD to promote weight gain through differential regulation of central energy metabolism pathways.

    May 09, 2017   doi: 10.1152/ajpendo.00369.2016   open full text
  • Roux-en-Y Gastric Bypass Surgery Enhances Contraction-Mediated Glucose Metabolism in Primary Human Myotubes.
    Hinkley, J. M., Zou, K., Park, S., Turner, K., Zheng, D., Houmard, J. A.
    AJP Endocrinology and Metabolism. May 09, 2017

    Contractile activity (e.g. exercise) evokes numerous metabolic adaptations in human skeletal muscle including enhanced insulin action and substrate oxidation. However, there is inter-subject variation in the physiological responses to exercise, which may be linked with factors such as the degree of obesity. Roux-en-Y gastric bypass (RYGB) surgery reduces body mass in severely obese (BMI > 40 kg/m2) individuals; however, it is uncertain whether RYGB can potentiate responses to contractile activity in this potentially exercise-resistant population. To examine possible interactions between RYGB and contractile activity, muscle biopsies were obtained from severely obese patients before and after RYGB, differentiated into myotubes, and electrically stimulated, after which changes in insulin action and glucose oxidation were determined. Prior to RYGB, myotubes were unresponsive to electrical stimulation as indicated by no changes in insulin-stimulated glycogen synthesis and basal glucose oxidation. However, myotubes from the same patients at 1 month after RYGB increased insulin-stimulated glycogen synthesis and basal glucose oxidation when subjected to contraction. While unresponsive before surgery, contraction improved insulin-stimulated phosphorylation of AS160 (Thr642, Ser704) after RYGB. These data suggest that RYGB surgery may enhance the ability of skeletal muscle from severely obese individuals to respond to contractile activity.

    May 09, 2017   doi: 10.1152/ajpendo.00413.2016   open full text
  • Cholestyramine treatment of healthy humans rapidly induces transient hypertriglyceridemia when treatment is initiated.
    Sjoberg, B., Straniero, S., Angelin, B., Rudling, M.
    AJP Endocrinology and Metabolism. May 09, 2017

    Bile acid (BA) production in mice is regulated by hepatic farnesoid X receptors and by intestinal fibroblast growth factor (FGF) 15, (in humans FGF19) a suppressor of BA synthesis that also reduces serum triglycerides and glucose. Cholestyramine treatment reduces FGF19 and induces BA synthesis while plasma triglycerides may increase of unclear reasons. We explored if FGF19 may suppress BA synthesis and plasma triglycerides in humans by modulation of FGF19 levels through long-term cholestyramine at increasing doses. In a second acute experiment, metabolic responses from one day of cholestyramine treatment were monitored. Long-term treatment reduced serum FGF19 by >90% and BA synthesis increased up to 17-fold while serum BAs, triglycerides, glucose and insulin were stable. After long-term treatment, serum BAs and FGF19 displayed rebound increases above baseline levels, and BA and cholesterol syntheses normalized after one week without rebound reductions. Acute cholestyramine treatment decreased FGF19 by 95% overnight and serum BAs by 60%, while BA synthesis increased 4-fold and triglycerides doubled. The results support that FGF19 represses BA synthesis but not serum triglycerides. However, after cessation of both long-term and one day cholestyramine treatment, circulating FGF19 levels were normalized within 2 days while BA synthesis remained significantly induced in both situations indicating that also other mechanisms than the FGF19 pathway are responsible for stimulation of BA synthesis elicited by cholestyramine. Several of the responses during cholestyramine treatment persisted at least 6 days after treatment highlighting the importance to remove such treatment at least a week prior to sampling.

    May 09, 2017   doi: 10.1152/ajpendo.00416.2016   open full text
  • Expression of microRNAs and target proteins in skeletal muscle of rats selectively bred for high and low running capacity.
    Pinto, S., Lamon, S., Stephenson, E. J., Kalanon, M., Mikovic, J., Koch, L. G., Britton, S. L., Hawley, J. A., Camera, D. M.
    AJP Endocrinology and Metabolism. May 02, 2017

    Abstract Impairments in mitochondrial function and substrate metabolism are implicated in the etiology of obesity and type 2 diabetes. MicroRNAs (miRNAs) can degrade mRNA or repress protein translation and have been implicated in the development of such disorders. We used a contrasting rat model system of selectively bred high- (HCR) or low- (LCR) intrinsic running capacity with established differences in metabolic health to investigate the molecular mechanisms through which miRNAs regulate target proteins mediating mitochondrial function and substrate oxidation processes. Quantification of select miRNAs using the Rat miFinder miRNA PCR array revealed differential expression of 15 skeletal muscle (m. tibialis anterior) miRNAs between HCR and LCR rats (14 with higher expression in LCR; P<0.05). Ingenuity Pathway Analysis predicted these altered miRNAs to collectively target multiple proteins implicated in mitochondrial dysfunction and energy substrate metabolism. Total protein abundance of citrate synthase (CS; miR-19 target) and voltage-dependent anion channel 1 (miR-7a target) were higher in HCR compared to LCR cohorts (~57 and ~26%, respectively; P<0.05). A negative correlation was observed for miR-19a-3p and CS (r =0.32, P=0.015) protein expression. To determine if miR-19a-3p can regulate CS in vitro we performed luciferase reporter and transfection assays in C2C12 myotubes. MiR-19a-3p binding to the CS untranslated region did not change luciferase reporter activity, however miR-19a-3p transfection decreased CS protein expression (~70%; P<0.05). The differential miRNA expression targeting proteins implicated in mitochondrial dysfunction and energy substrate metabolism may contribute to the molecular basis mediating the divergent metabolic health profiles of LCR and HCR rats.

    May 02, 2017   doi: 10.1152/ajpendo.00043.2017   open full text
  • Development of diabetes does not alter behavioral and molecular circadian rhythms in a transgenic rat model of Type 2 Diabetes Mellitus.
    Qian, J., Thomas, A. P., Schroeder, A. M., Rakshit, K., Colwell, C. S., Matveyenko, A. V.
    AJP Endocrinology and Metabolism. May 02, 2017

    Metabolic state and circadian clock function exhibit complex bidirectional relationship. Circadian disruption increases propensity for metabolic dysfunction, whereas common metabolic disorders such as obesity and Type 2 diabetes (T2DM) are associated with impaired circadian rhythms. Specifically, alterations in glucose availability and glucose metabolism have been shown to modulate clock gene expression and function in vitro; however to date it is unknown whether development of diabetes imparts deleterious effects on the suprachiasmatic nucleus (SCN) circadian clock and SCN-driven outputs in vivo. To address this question, we undertook studies in aged diabetic rats transgenic for human islet amyloid polypeptide (h-IAPP), an established non-obese model of T2DM (HIP rat) which develops metabolic defects closely recapitulating those present in patients with T2DM. HIP rats were also crossbred with a clock gene reporter rat model (Per1:luciferase transgenic rat) to permit assessment of the SCN and the peripheral molecular clock function ex-vivo. Utilizing these animal models, we examined effects of diabetes on 1) behavioral circadian rhythms, 2) photic entrainment of circadian activity, 3) SCN and peripheral tissue molecular clock function and 4) regulation of melatonin secretion. We report that circadian activity, light-induced entrainment, molecular clockwork, as well as melatonin secretion is preserved in the HIP rat model of T2DM. These results suggest that despite well-characterized ability of glucose to acutely modulate circadian clock gene expression in vitro, SCN clock function and key behavioral and physiological outputs appear to be preserved under chronic diabetic conditions characteristic of non-obese T2DM.

    May 02, 2017   doi: 10.1152/ajpendo.00406.2016   open full text
  • Low dose leptin infusion in the fourth ventricle of rats enhances the response to third ventricle leptin injection.
    Harris, R. B. S.
    AJP Endocrinology and Metabolism. April 25, 2017

    We previously reported that low dose leptin infusions into the 3rd or 4th ventricle that do not effect energy balance when given independently cause rapid weight loss when given simultaneously. Therefore, we tested whether hindbrain leptin enhances the response to forebrain leptin, or whether forebrain leptin enhances the response to hindbrain leptin. Rats received 4th ventricle infusions of saline, 0.01, 0.1, 0.3 or 0.6 μg leptin/day for 13 days. On days 9 and 13 0.1 μg leptin was injected into the 3rd ventricle. The injection inhibited food intake for 36 hours in saline infused rats, but for 60 hours in those infused with 0.6 μg leptin/day. Leptin injection increased IBAT temperature in leptin infused, but not saline-infused rats. In a separate experiment rats received 3rd ventricle infusions of saline, 0.005, 0.01, 0.05 or 0.1 μg leptin/day and 4th ventricle injections of 1.0 μg leptin on days 9 and 13. Leptin injection inhibited food intake, RER and 14 hour food intake in rats infused with saline or the two lowest doses of leptin. There was no effect with higher dose leptin infusions because food intake, body fat and lean mass were already inhibited. These data suggest that activation of leptin receptors in the hindbrain enhances the response to 3rd ventricle leptin, whereas activation of forebrain leptin receptors does not enhance the response to 4th ventricle leptin, consistent with our previous finding that weight loss in rats treated with 4th ventricle leptin is associated with indirect activation of hypothalamic STAT3.

    April 25, 2017   doi: 10.1152/ajpendo.00052.2017   open full text
  • Changes in LXR signaling influence early-pregnancy lipogenesis and protect against dysregulated fetoplacental lipid homeostasis.
    Nikolova, V., Papacleovoulou, G., Bellafante, E., Borges Manna, L., Jansen, E., Baron, S., Abu-Hayyeh, S., Parker, M. G., Williamson, C.
    AJP Endocrinology and Metabolism. April 18, 2017

    Human pregnancy is associated with enhanced de novo lipogenesis in the early stages followed by hyperlipidemia during advanced gestation. Liver X receptors (LXRs) are oxysterol-activated nuclear receptors which stimulate de novo lipogenesis and also promote the efflux of cholesterol from extrahepatic tissues followed by its transport back to the liver for biliary excretion. Although LXR is recognized as a master regulator of triglyceride and cholesterol homeostasis it is unknown whether it facilitates the gestational adaptations in lipid metabolism. To address this question, biochemical profiling, protein quantification and gene expression studies were used, and gestational metabolic changes in T0901317-treated wild-type mice and LXRα, β-/- mutants were investigated. Here, we show that altered LXR signaling contributes to the enhanced lipogenesis in early pregnancy by increasing the expression of hepatic Fas and stearoyl-CoA desaturase 1 (Scd1). Both the pharmacological activation of LXR with T0901317 and the genetic ablation of its two isoforms disrupted the increase in hepatic fatty acid biosynthesis and the development of hypertriglyceridemia during early gestation. We also demonstrate that absence of LXR enhances maternal white adipose tissue lipolysis, causing abnormal accumulation of triglycerides, cholesterol and free fatty acids in the fetal liver. Together, these data identify LXR as an important factor in early-pregnancy lipogenesis which is also necessary to protect against abnormalities in fetoplacental lipid homeostasis.

    April 18, 2017   doi: 10.1152/ajpendo.00449.2016   open full text
  • Fatty acid uptake and blood flow in adipose tissue compartments of morbidly obese subjects with or without type 2 diabetes: effects of bariatric surgery.
    Dadson, P., Ferrannini, E., Landini, L., Hannukainen, J. C., Kalliokoski, K. K., Vaittinen, M., Honka, H., Karlsson, H. K., Tuulari, J. J., Soinio, M., Salminen, P., Parkkola, R., Pihlajamaki, J., Iozzo, P., Nuutila, P.
    AJP Endocrinology and Metabolism. April 11, 2017

    Body fat accumulation, distribution and metabolic activity are factors in the pathophysiology of obesity and type 2 diabetes (T2D). We investigated adipose blood flow, fatty acid uptake (FAU), and subcutaneous and visceral fat cellularity in obese patients with or without T2D. A total 23 morbidly obese (BMI 42 kg/m2) patients were studied before and 6 months after bariatric surgery; 15 nonobese subjects served as controls. Positron emission tomography was used to measure tissue FAU (with 18F-FTHA) and blood flow (with H215O); MRI was used for fat distribution, fat biopsy for adipocyte size. Obese subjects had subcutaneous hyperplasia and hypertrophy, and lower blood flow; when expressed per cell, flow was similar to controls. FAU into subcutaneous and visceral depots was increased in the obese; per unit tissue mass, however, FAU was similar to controls but reduced in skeletal muscle. Fatty acid fractional extraction in subcutaneous fat and muscle was only increased in obese patients with T2D. We conclude that surgery leads to metabolic improvement and reduces subcutaneous fat hyperplasia and hypertrophy; subcutaneous blood flow and FAU decrease in absolute terms and per cell while fractional FAU remains unchanged in T2D. In the obese, subcutaneous blood flow is a determinant of FAU and is coupled with cellularity; efficiency of FAU is enhanced in subcutaneous fat and muscle in T2D.

    April 11, 2017   doi: 10.1152/ajpendo.00044.2017   open full text
  • Western-diet consumption induces alteration of barrier function mechanisms in the ileum that correlates with metabolic endotoxemia in rats.
    Guerville, M., Leroy, A., Sinquin, A., Laugerette, F., Michalski, M.-C., Boudry, G.
    AJP Endocrinology and Metabolism. April 11, 2017

    Obesity and its related disorders have been associated to the presence in the blood of gut bacteria-derived lipopolysaccharides (LPS). However, the factors underlying this low-grade elevation in plasma LPS, so-called metabolic endotoxemia, are not fully elucidated. We aimed to investigate the effects of Western diet (WD) feeding on intestinal and hepatic LPS handling mechanisms in a rat model of diet-induced obesity (DIO). Rats were fed either a standard chow diet (C) or a Western Diet (WD, 45% fat) for 6 weeks. They were either fed ad libitum or pair-fed to match the caloric intake of Crats for the first week then fed ad libitum for the remaining 5 weeks. Six-week WD feeding led to a mild obese phenotype with increased adiposity and elevated serum LPS-binding protein (LBP) levels relative to C rats, irrespective of initial energy intake. Serum LPS was not different between dietary groups but exhibited strong variability. Disrupted ileal mucus secretion and decreased ileal Reg3- and -β gene expression along with high ileal permeability to LPS were observed in WD compared to C-fed rats. Ileal and caecal intestinal alkaline phosphatase (IAP) activity as well as Verrucomicrobia and Bifidobacterium caecal levels were increased in WD-fed rats compared to C-fed rats. WD consumption did not impact mRNA levels of LPS-handling hepatic enzymes. Correlation analysis revealed that ileal passage of LPS, IAP activity, Proteobacteria levels and hepatic aoah gene expression correlated with serum LPS and LBP, suggesting that ileal mucosal defense impairment induced by WD feeding contribute to metabolic endotoxemia.

    April 11, 2017   doi: 10.1152/ajpendo.00372.2016   open full text
  • Cyp8b1 ablation prevents western diet-induced weight gain and hepatic steatosis due to impaired fat absorption.
    Bertaggia, E., Jensen, K. K., Castro-Perez, J., Xu, Y., Di Paolo, G., Chan, R. B., Wang, L., Haeusler, R. A.
    AJP Endocrinology and Metabolism. April 04, 2017

    Bile acids (BAs) are cholesterol derivatives that regulate lipid metabolism, through their dual abilities to promote lipid absorption and activate BA receptors. However, different BA species have varying abilities to perform these functions. Eliminating 12α-hydroxy BAs in mice via Cyp8b1 knockout causes low body weight and improved glucose tolerance. The goal of this study was to determine mechanisms of low body weight in Cyp8b1-/- mice. We challenged Cyp8b1-/- mice with western type diet and assessed body weight and composition. We measured energy expenditure, fecal calories, lipid absorption and performed lipidomic studies on feces and intestine. We investigated the requirement for dietary fat in the phenotype using a fat-free diet. Cyp8b1-/- mice were resistant to western diet-induced body weight gain, hepatic steatosis, and insulin resistance. These changes were associated with increased fecal calories, due to malabsorption of hydrolyzed dietary triglycerides. This was reversed by treating the mice with taurocholic acid, the major 12α-hydroxylated BA species. The improvements in body weight and steatosis were normalized by feeding mice a fat-free diet. The effects of BA composition on intestinal lipid handling are important for whole-body energy homeostasis. Thus, modulating BA composition is a potential tool for obesity or diabetes therapy.

    April 04, 2017   doi: 10.1152/ajpendo.00409.2016   open full text
  • Gonad-related factors promote muscle performance gain during postnatal development in male and female mice.
    Ueberschlag-Pitiot, V., Stantzou, A., Messeant, J., Lemaitre, M., Owens, D. J., Noirez, P., Roy, P., Agbulut, O., Metzger, D., Ferry, A.
    AJP Endocrinology and Metabolism. March 28, 2017

    In order to better define the role of male and female gonad-related factors (MGRF, presumably testosterone, and FGRF, presumably estradiol, respectively) on mouse hindlimb skeletal muscle contractile performance/function gain during postnatal development, we analysed the effect of castration initiated before puberty in male and female mice. We found that muscle absolute and specific (normalized to muscle weight) maximal forces were decreased in 6-month old male and female castrated mice, as compared to age- and sex-matched intact mice, without alteration in neuromuscular transmission. Moreover, castration decreased absolute and specific maximal powers, another important aspect of muscle performance, in 6-month old males, but not in females. Absolute maximal force was similarly reduced by castration in 3-month old muscle fibre androgen receptor (AR) -deficient and wild-type male mice, indicating that the effect of MGRF was muscle fibre AR independent. Castration reduced the muscle weight gain in 3-month mice of both sexes and in 6-month females but not in males. We also found that bone morphogenetic protein signaling through Smad1/5/9 was not altered by castration in atrophic muscle of 3-month old mice of both sexes. Moreover, castration decreased the sexual dimorphism regarding muscle performance. Together these results demonstrated that in the long-term MGRF and FGRF promote muscle performance gain in mice during postnatal development, independently of muscle growth in males, largely via improving muscle contractile quality (force and power normalized) and that MGFR and FGRF also contribute to sexual dimorphism. However, the mechanisms underlying MGFR and FGRF actions remain to be determined

    March 28, 2017   doi: 10.1152/ajpendo.00446.2016   open full text
  • Glucose oxidation positively regulates glucose uptake and improves cardiac function recovery after myocardial reperfusion.
    Li, T., Xu, J., Qin, X., Hou, Z., Guo, Y., Liu, Z., Wu, J., Zheng, H., Zhang, X., Gao, F.
    AJP Endocrinology and Metabolism. March 21, 2017

    Myocardial reperfusion decreases glucose oxidation and uncouples glucose oxidation from glycolysis. Therapies that increase glucose oxidation lessen myocardial ischemia/reperfusion injury. However, the regulation of glucose uptake during reperfusion remains poorly understood. Here we found that glucose uptake was remarkably diminished in myocardium following reperfusion in Sprague-Dawley rats as detected by 18F-labeled and fluorescent-labeled glucose analogs, even though GLUT1 was upregulated by 3 folds and GLUT4 translocation remained unchanged compared with those of sham rats. The decreased glucose uptake was accompanied by suppressed glucose oxidation. Interestingly, stimulating glucose oxidation by inhibition of pyruvate dehydrogenase kinase 4 (PDK4), a rate-limiting enzyme for glucose oxidation, increased glucose uptake and alleviated ischemia/reperfusion injury. In vitro data in neonatal myocytes showed that PDK4 overexpression decreased glucose uptake, while its knockdown increased glucose uptake, suggesting a role of PDK4 in regulating glucose uptake. Moreover, inhibition of PDK4 increased myocardial glucose uptake with concomitant enhancement of cardiac insulin sensitivity following myocardial ischemia/reperfusion. These results showed that the suppressed glucose oxidation mediated by PDK4 contributes to the reduced glucose uptake in myocardium following reperfusion, and enhancement of glucose uptake exerts cardioprotection. The findings suggest that stimulating glucose oxidation via PDK4 could be an efficient approach to improve recovery from myocardial ischemia/reperfusion injury.

    March 21, 2017   doi: 10.1152/ajpendo.00014.2017   open full text
  • Effects of genetic deletion of soluble 5'-nucleotidases NT5C1A and NT5C2 on AMPK activation and nucleotide levels in contracting mouse skeletal muscles.
    Kviklyte, S., Vertommen, D., Yerna, X., Andersen, H., Xu, X., Gailly, P., Bohlooly-Y, M., Oscarsson, J., Rider, M. H.
    AJP Endocrinology and Metabolism. March 21, 2017

    AMP-activated protein kinase (AMPK) plays a key role in energy homeostasis and is activated in response to contraction-induced ATP depletion in skeletal muscle via a rise in intracellular AMP/ADP concentrations. AMP can be deaminated by AMP-deaminase to IMP, which is hydrolysed to inosine by cytosolic 5'-nucleotidase-II (NT5C2). AMP can also be hydrolysed to adenosine by cytosolic 5'-nucleotidase-IA (NT5C1A). Previous gene silencing and overexpression studies indicated control of AMPK activation by NT5C enzymes. In the present study using gene knockout mouse models, we investigated effects of NT5C1A and NT5C2 deletion on intracellular adenine nucleotide levels and AMPK activation in electrically stimulated skeletal muscles. Surprisingly, NT5C enzyme knockout did not lead to enhanced AMP or ADP concentrations in response to contraction, with no potentiation of increases in AMPK activity in extensor digitorum longus (EDL) and soleus mouse muscles. Moreover, dual blockade of AMP metabolism in EDL using an AMPD inhibitor combined with NT5C1A deletion did not enhance rises in AMP and ADP or increased AMPK activation by electrical stimulation. The results on muscles from the NT5C knockout mice contradict previous findings where AMP levels and AMPK activity were shown to be modulated by NT5C enzymes.

    March 21, 2017   doi: 10.1152/ajpendo.00304.2016   open full text
  • Whey protein-effects on energy balance link the intestinal mechanisms of energy absorption with adiposity and hypothalamic neuropeptide gene expression.
    Nilaweera, K. N., Cabrera-Rubio, R., Speakman, J. R., O' Connor, P. M., McAuliffe, A., Guinane, C. M., Lawton, E., Crispie, F., Aguilera, M., Stanley, M., Boscaini, S., Joyce, S., Melgar, S., Cryan, J. F., Cotter, P. D.
    AJP Endocrinology and Metabolism. March 21, 2017

    We tested the hypothesis that dietary whey protein isolate (WPI) affects the intestinal mechanisms related to energy absorption and that the resulting energy deficit is compensated by changes in energy balance to support growth. C57BL/6 mice were provided a diet enriched with WPI with varied sucrose content, and the impact on energy balance related parameters were investigated. As part of a high sucrose diet, WPI reduced the hypothalamic expression of pro-opiomelanocortin gene expression and increased energy intake. The energy expenditure was unaffected, but epididymal weight was reduced, indicating an energy loss. Notably, there was a reduction in the ileum gene expression for amino acid transporter SLC6a19, glucose transporter 2 and fatty acid transporter 4. The composition of the gut microbiota also changed, where Firmicutes were reduced. The above changes indicated a reduced energy absorption through the intestine. We propose that this mobilised energy in the adipose tissue and caused hypothalamic changes that increased energy intake, acting to counteract the energy deficit arising in the intestine. Lowering the sucrose content in the WPI diet increased energy expenditure. This further reduced epididymal weight and plasma leptin, whereupon hypothalamic ghrelin gene expression and the intestinal weight were both increased. These data suggest that when the intestine-adipose-hypothalamic pathway is subjected to an additional energy loss (now in the adipose tissue), compensatory changes attempt to assimilate more energy. Notably, WPI and sucrose content interact to enable the component mechanisms of this pathway.

    March 21, 2017   doi: 10.1152/ajpendo.00356.2016   open full text
  • Overactivation of the endocannabinoid system alters the anti-lipolytic action of insulin in mouse adipose tissue.
    Muller, T., Demizieux, L., Troy-Fioramonti, S., Gresti, J., Pais de Barros, J.-P., Berger, H., Verges, B., Degrace, P.
    AJP Endocrinology and Metabolism. March 21, 2017

    Evidence has accumulated that obesity-related metabolic dysregulation is associated with overactivation of the endocannabinoid system (ECS), which involves cannabinoid receptor 1 (CB1R), in peripheral tissues, including adipose tissue (AT). The functional consequences of CB1R activation on AT metabolism remain unclear. Since excess fat mobilization is considered an important primary event contributing to the onset of insulin resistance, we combined in vivo and in vitro experiments to investigate whether activation of ECS could alter the lipolytic rate. For this purpose, the appearance of plasma glycerol was measured in wild-type and CB1R-/- mice after acute anandamide administration or inhibition of endocannabinoid degradation by JZL195. Additional experiments were conducted on rat AT explants to evaluate the direct consequences of ECS activation on glycerol release and signaling pathways. Treatments stimulated glycerol release in mice fasted for 6 h and injected with glucose but not in 24-h fasted mice or in CB1R-/- suggesting that the effect was dependent on plasma insulin levels and mediated by CB1R. We concomitantly observed that Akt cascade activity was decreased, indicating an alteration of the anti-lipolytic action of insulin. Similar results were obtained with tissue explants exposed to anandamide, thus identifying CB1R of AT as a major target. This study indicates the existence of a functional interaction between CB1R and lipolysis regulation in AT. Further investigation is needed to test whether the elevation of ECS tone encountered in obesity is associated with excess fat mobilization contributing to ectopic fat deposition and related metabolic disorders.

    March 21, 2017   doi: 10.1152/ajpendo.00374.2016   open full text
  • Does maternal-fetal transfer of creatine occur in pregnant sheep?
    Baharom, S., De Matteo, R., Ellery, S., Della Gatta, P., Bruce, C. R., Kowalski, G. M., Hale, N., Dickinson, H., Harding, R., Walker, D., Snow, R. J.
    AJP Endocrinology and Metabolism. March 21, 2017

    Our aim was to determine the disposition of creatine in ovine pregnancy, and whether creatine is transferred across the placenta from mother to fetus. Pregnant ewes received either: (i) a continuous intravenous infusion of creatine monohydrate or saline from 122 to 131 days gestation, with maternal and fetal arterial blood and amniotic fluid samples collected daily for creatine analysis and fetal tissues collected at necropsy at 133 days for analysis of creatine content, or; (ii) a single systemic bolus injection of 13C labelled creatine monohydrate at 130 days of gestation, with maternal and fetal arterial blood, uterine vein blood and amniotic fluid samples collected prior to, and for 4 hours post injection and analysed for creatine, creatine isotopic enrichment, and guanidinoacetic acid (GAA, precursor of creatine) concentrations. Presence of the creatine transporter-1 (SLC6A8), and arginine-glycine amidinotransferase (AGAT, the enzyme synthesising GAA) proteins were determined by western blots of placental cotyledons. The 10-day creatine infusion increased maternal plasma creatine concentration 3-4 fold (P<0.05), without significantly changing fetal arterial, amniotic fluid, fetal tissues, or placental creatine content. Maternal arterial 13C enrichment was increased (P<0.05) post bolus 13C-creatine injection, without change of fetal arterial 13C enrichment. SLC6A8 and AGAT proteins were identified in placental cotyledons, and GAA concentration was significantly higher in uterine vein than maternal artery plasma. Despite the presence of SLC6A8 protein in cotyledons, these results suggest that creatine is not transferred from mother to fetus in near-term sheep, and that the ovine utero-placental unit releases GAA into the maternal circulation.

    March 21, 2017   doi: 10.1152/ajpendo.00450.2016   open full text
  • Feeding Influences Adipose Tissue Responses To Exercise In Overweight Men.
    Chen, Y.-C., Travers, R. L., Walhin, J.-P., Gonzalez, J. T., Koumanov, F., Betts, J. A., Thompson, D.
    AJP Endocrinology and Metabolism. March 14, 2017

    Feeding profoundly affects metabolic responses to exercise in various tissues but the effect of feeding status on human adipose tissue responses to exercise has never been studied. Ten healthy overweight men aged 26 ± 5 years (mean ± SD) with a waist circumference of 105 ± 10 cm walked at 60% of maximum oxygen uptake under either FASTED or FED conditions in a randomised, counterbalanced design. Feeding comprised 648 ± 115 kcal 2 h before exercise. Blood samples were collected at regular intervals to examine changes in metabolic parameters and adipokine concentrations. Adipose tissue samples were obtained at baseline and one hour post-exercise to examine changes in adipose tissue mRNA expression and secretion of selected adipokines ex-vivo. Adipose tissue mRNA expression of PDK4, ATGL, HSL, FAT/CD36, GLUT4 and IRS2 in response to exercise were lower in FED compared to FASTED conditions (all p ≤ 0.05). Post-exercise adipose IRS2 protein was affected by feeding (p ≤ 0.05), but Akt2, AMPK, IRS1, GLUT4, PDK4 and HSL protein levels were not different. Feeding status did not impact serum and ex-vivo adipose secretion of IL-6, leptin or adiponectin in response to exercise. This is the first study to show that feeding prior to acute exercise affects post-exercise adipose tissue gene expression and we propose that feeding is likely to blunt long-term adipose tissue adaptation to regular exercise.

    March 14, 2017   doi: 10.1152/ajpendo.00006.2017   open full text
  • Ovarian Function's Role During Cancer Cachexia Progression in the Female Mouse.
    Hetzler, K. L., Hardee, J. P., LaVoie, H. A., Murphy, E. A., Carson, J. A.
    AJP Endocrinology and Metabolism. March 14, 2017

    Cachexia is a debilitating condition that occurs with chronic disease including cancer; our research has shown that some regulation of cancer cachexia progression is affected by sex. The ApcMin/+ mouse is genetically predisposed to develop intestinal tumors; interleukin-6 (IL-6) signaling and hypogonadism are associated with cachexia severity in the male. This relationship in the female warrants further investigation, as we have shown that the ability of IL-6 to induce cachexia differs between the sexes. Since ovarian reproductive function relies on a complex system of endocrine signaling to affect whole body homeostasis, we examined the relationship between ovarian reproductive function and the progression of cancer cachexia in the female ApcMin/+ mouse. Ovarian reproductive function was monitored in female ApcMin/+ mice. Disease-related cessation of estrous cycling (acyclicity) was seen in 38% of mice. Acyclicity was associated with severe cachexia including morphological and functional losses and enhanced muscle inflammatory gene expression. Interestingly, ovariectomy rescued body weight, muscle mass and function, but increased muscle sensitivity to systemic IL-6 overexpression. In conclusion, our results provide evidence for a relationship between ovarian reproduction function and cachexia progression in female ApcMin/+ mice.

    March 14, 2017   doi: 10.1152/ajpendo.00294.2016   open full text
  • Phenylalanine isotope pulse method to measure effect of sepsis on protein breakdown and membrane transport in the pig.
    Ten Have, G. A. M., Engelen, M. P., Wolfe, R. R., Deutz, N. E. P.
    AJP Endocrinology and Metabolism. March 14, 2017

    The primed-continuous (PC) phenylalanine (Phe) stable isotope infusion methodology is often used as a proxy for measuring whole body protein breakdown (WbPB) in sepsis. It is unclear, if WbPB data obtained by an easy-to-use single IV Phe isotope pulse administration (PULSE) are comparable to those by PC. Compartmental modeling with PULSE could provide us more insight in WbPB in sepsis. Therefore, in the present study, we compared PULSE with PC as proxy for WbPB in an instrumented pig model with Pseudomonas aeruginosa induced severe sepsis (Healthy: n=9; Sepsis: n=13). Seventeen hours after sepsis induction, we compared the Wb rate of appearance (WbRa) of Phe obtained by PC(L-[15N]-Phe) and PULSE(L-[ring-13C6]-Phe) in arterial plasma using LC-MS/MS and (non)compartmental modeling. PULSE-WbRa was highly correlated with PC-WbRa (r=0.732, p<0.0001) and WbPB (r=0.897, p<0.0001) independent of the septic state. PULSE-WbRa was 1.6 times higher than PC-WbRa (p<0.001). Compartmental and non-compartmental PULSE modeling provides comparable WbRa values, although compartmental modeling was more sensitive. WbPB was elevated in sepsis (Healthy: 3378±103; Sepsis: 4333±160 nmol/kg BW/min, p=0.0002). With PULSE, sepsis was characterized by an increase of the metabolic shunting (Healthy: 3021±347; Sepsis: 4233±344 nmol/kg BW/min, p=0.026). Membrane transport capacity was the same. Both PC and PULSE-methods are able to assess changes in WbRa of plasma Phe reflecting WbPB changes with high sensitivity, independent of the (patho-)physiological state. The easy-to-use (non-)compartmental PULSE reflects better the real WbPB than PC. With PULSE compartmental analysis, we conclude that the membrane transport capacity for amino acids is not compromised in severe sepsis.

    March 14, 2017   doi: 10.1152/ajpendo.00351.2016   open full text
  • Optimization of Tolerability and Efficacy of Dual Amylin and Calcitonin Receptor Agonist, KBP-089, through Dose Escalation and Combination with a GLP-1 Analogue.
    Gydesen, S., Andreassen, K. V., Hjuler, S. T., Hellgren, L. I., Karsdal, M. A., Henriksen, K.
    AJP Endocrinology and Metabolism. March 14, 2017

    Amylin and GLP-1 agonism induce a well-known anorexic effect at dose initiation, which is managed by dose escalation. In this study, we investigated how to optimize tolerability, while maintaining efficacy of KBP-089. Furthermore, we tested the GLP-1 add-on potential of KBP-089 in HFD rats. KBP-089 potently activated both the amylin and calcitonin receptors in vitro, demonstrated a prolonged receptor activation, and potently reduced acute food intake. HFD rats dosed every or every second day obtained equal weight loss at study end, albeit with an uneven reduction in both food intake and bodyweight in rats dosed every second day. In a 4-fold dose escalation, KBP-089 transiently reduced food intake at every escalation step - with reducing magnitude over time - and the following treatment with 2.5, 10 and 40 µg/kg resulted in a ~15% vehicle corrected weight loss, a corresponding reduction in adipose tissue (AT), and all treatment groups improved oral glucose tolerance (p<0.01). Two-fold and linear escalations suppressed bodyweight evenly with no significant reduction in food intake at either escalation step. KBP-089 (1.25 µg/kg) and liraglutide (50 µg/kg) lowered bodyweight 8% and 2% in HFD rats, respectively, while the combination resulted in a 12% bodyweight reduction. Moreover, the combination improved glucose tolerance (p<0.05) In conclusion, DACRAs act complementary with GLP-1 on food intake and bodyweight. Furthermore, upon escalation KBP-089 was well tolerated, induced and sustained a significant weight loss and a reduction in AT in lean and HFD rats, underscoring the potential of KBP-089 as an anti-obesity agent.

    March 14, 2017   doi: 10.1152/ajpendo.00419.2016   open full text
  • GPR40 reduces food intake and body weight through GLP-1.
    Gorski, J. N., Pachanski, M. J., Mane, J., Plummer, C. W., Souza, S., Thomas-Fowlkes, B. S., Ogawa, A. M., Weinglass, A. B., Di Salvo, J., Cheewatrakoolpong, B., Howard, A. D., Colletti, S. L., Trujillo, M. E.
    AJP Endocrinology and Metabolism. March 14, 2017

    GPR40 partial agonists lower glucose through the potentiation of glucose-stimulated insulin secretion, which is believed to provide significant glucose lowering without the weight gain or hypoglycemic risk associated with exogenous insulin or glucose independent insulin secretagogues. The class of small molecule GPR40 modulators, known as AgoPAMs (agonist also capable of acting as positive allosteric modulators), differentiate from partial agonists, binding to a distinct site and functioning as full agonists to stimulate the secretion of both insulin and GLP-1 (17). Here we show that GPR40 AgoPAM's significantly increase active GLP-1 levels and reduce acute and chronic food intake and body weight in diet-induced obese (DIO) mice. These effects of AgoPAM treatment on food intake are novel and required both GPR40 and GLP-1 receptor signaling pathways, as demonstrated in GPR40 and GLP1 receptor-null mice. Further, weight loss associated with GPR40 AgoPAMs was accompanied by a significant reduction in gastric motility in these DIO mice. Chronic treatment with a GPR40 AgoPAM, in combination with a dipeptidyl peptidase-IV (DPP-IV) inhibitor, synergistically decreased food intake and body weight in the mouse. The effect of GPR40 AgoPAMs on GLP-1 secretion was recapitulated in lean, healthy Rhesus macaque demonstrating the putative mechanism mediating weight loss translates to higher species. Together, our data indicate effects of AgoPAMs that go beyond glucose lowering previously observed with GPR40 partial agonist treatment with additional potential for weight loss.

    March 14, 2017   doi: 10.1152/ajpendo.00435.2016   open full text
  • Oxygen delivery and the restoration of the muscle energetic balance following exercise: Implications for delayed muscle recovery in patients with COPD.
    Layec, G., Hart, C. R., Trinity, J. D., Kwon, O.-S., Rossman, M. J., Broxterman, R. M., Le Fur, Y., Jeong, E.-K., Richardson, R. S.
    AJP Endocrinology and Metabolism. March 14, 2017

    Patients with chronic obstructive pulmonary disease (COPD) experience a delayed recovery from skeletal muscle fatigue following exhaustive exercise that likely contributes to their progressive loss of mobility. As this phenomenon is not well understood, this study sought to examine post-exercise peripheral oxygen (O2) transport and muscle metabolism dynamics in patients with COPD, two important determinants of muscle recovery. Twenty four subjects, 12 non-hypoxemic patients with COPD and 12 healthy subjects with a sedentary lifestyle, performed dynamic plantar flexion exercise at 40% of maximal work rate (WRmax) with phosphorus magnetic resonance spectroscopy (31P-MRS), near-infrared spectroscopy (NIRS), and vascular Doppler ultrasound assessments. The mean response time of limb blood flow at the offset of exercise was significantly prolonged in patients with COPD (Controls:56±27s; COPD:120±87s; P<0.05). In contrast, the post-exercise time constant for capillary blood flow was not significantly different between groups (Controls:49±23s; COPD:51±21s; P>0.05). The initial post-exercise convective O2 delivery (Controls:0.15±0.06 L.min-1; COPD:0.15±0.06 L.min-1) and the corresponding oxidative adenosine triphosphate (ATP) demand (Controls: 14±6 mM.min-1; COPD: 14±6 mM.min-1) in the calf were not significantly different between controls and patients with COPD (P>0.05). The PCr resynthesis time constant (Controls:46±20 s; COPD:49±21 s), peak mitochondrial phosphorylation rate, and initial proton efflux were also not significantly different between groups (P>0.05). Therefore, despite perturbed peripheral hemodynamics, intracellular O2 availability, proton efflux, and aerobic metabolism recovery in the skeletal muscle of non-hypoxemic patients with COPD are preserved following plantar flexion exercise and, thus, are unlikely to contribute to the delayed recovery from exercise in this population.

    March 14, 2017   doi: 10.1152/ajpendo.00462.2016   open full text
  • Menin and PRMT5 suppress GLP1 receptor transcript and PKA-mediated phosphorylation of FOXO1 and CREB.
    Muhammad, A. B., Xing, B., Liu, C., Naji, A., Ma, X., Simmons, R. A., Hua, X.
    AJP Endocrinology and Metabolism. March 07, 2017

    Menin is a scaffold protein that interacts with several epigenetic mediators to regulate gene transcription, and suppresses pancreatic beta cell proliferation. Tamoxifen inducible deletion of multiple endocrine neoplasia type 1 (MEN1) gene, which encodes the protein menin, increases beta cell mass in multiple murine models of diabetes and ameliorates diabetes. Glucagon-like-peptide-1 (GLP1) is another key physiological modulator of beta cell mass and glucose homeostasis. However, it is not clearly understood whether menin crosstalks with GLP1 signaling. Here we show that menin and protein arginine methyltransferase 5 (PRMT5) suppress GLP1 receptor (GLP1R) transcript levels. Notably, a GLP1R agonist induces phosphorylation of forkhead box protein O1 (FOXO1) at S253, and the phosphorylation is mediated by protein kinase A (PKA). Interestingly, menin suppresses GLP1-induced and PKA-mediated phosphorylation of both FOXO1 and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), likely through a protein arginine methyltransferase. Menin mediated suppression of FOXO1 and CREB phosphorylation increases FOXO1 levels and suppresses CREB target genes, respectively. A small molecule menin inhibitor reverses menin-mediated suppression of both FOXO1 and CREB phosphorylation. In addition, ex vivo treatment of both mouse and human pancreatic islets with a menin inhibitor increases levels of proliferation marker Ki67. In conclusion, our results suggest that menin and PRMT5 suppress GLP1R transcript levels and PKA-mediated phosphorylation of FOXO1 and CREB, and a menin inhibitor may reverse this suppression to induce beta cell proliferation.

    March 07, 2017   doi: 10.1152/ajpendo.00241.2016   open full text
  • Comprehensive functional screening of miRNAs involved in fat cell insulin sensitivity among women.
    Dahlman, I., Belarbi, Y., Laurencikiene, J., Pettersson, A. M., Arner, P., Kulyte, A.
    AJP Endocrinology and Metabolism. March 07, 2017

    The key pathological link between obesity and type 2 diabetes is insulin resistance but the molecular mechanisms are not entirely identified. MicroRNAs (miRNA) are dysregulated in obesity and may contribute to insulin resistance. Our objective was to detect and functionally investigate miRNAs linked to insulin sensitivity in human subcutaneous white adipose tissue (scWAT). Subjects were selected based on the insulin-stimulated lipogenesis response of subcutaneous adipocytes. Global miRNA profiling was performed in abdominal scWAT of 18 obese insulin resistance (OIR), 21 obese insulin sensitive (OIS) and nine lean women. MicroRNAs demonstrating differential expression between OIR and OIS women were overexpressed in human in vitro-differentiated adipocytes followed by assessment of lipogenesis and identification of miRNA targets by measuring mRNA/protein expression and 3'UTR analysis. Eleven miRNAs displayed differential expression between OIR and OIS states. Overexpression of miR-143-3p and miR-652-3p increased insulin-stimulated lipogenesis in human in vitrodifferentiated adipocytes and directly or indirectly affected several genes/proteins involved in insulin signaling at transcriptional or post-transcriptional levels. Adipose expression of miR-143-3p and miR-652-3p was positively associated with insulin-stimulated lipogenesis in scWAT independently of BMI. In conclusion, miR-143-3p and miR-652-3p are linked to scWAT insulin resistance independently of obesity and influence insulin-stimulated lipogenesis by interacting at different steps with insulin signaling pathways.

    March 07, 2017   doi: 10.1152/ajpendo.00251.2016   open full text
  • Liver X receptor {alpha} induces 17{beta}-hydroxysteroid dehydrogenase-13 expression through SREBP-1c.
    Su, W., Peng, J., Li, S., Dai, Y.-b., Wang, C.-j., Xu, H., Gao, M., Ruan, X.-z., Gustafsson, J.-A., Zhang, X.-y., Guan, Y.-f.
    AJP Endocrinology and Metabolism. March 07, 2017

    Liver X receptors, including LXRα and LXRβ, are known to be master regulators of liver lipid metabolism. Activation of LXRα increases hepatic lipid storage in lipid droplets (LDs). 17β-hydroxysteroid dehydrogenase-13 (17β-HSD13), a recently identified liver-specific LD-associated protein, has been reported to be involved in the development of nonalcoholic fatty liver disease. However, little is known about its transcriptional regulation. In the present study, we aimed at determining whether 17β-HSD13 gene transcription is controlled by LXRs. We found that treatment with T0901317, a non-specific LXR agonist, increased both 17β-HSD13 mRNA and protein levels in cultured hepatocytes. It also significantly upregulated hepatic 17β-HSD13 expression in wild-type (WT) and LXRβ-/- mice but not in LXRα-/- mice. Basal expression of 17β-HSD13 in the livers of LXRα-/- mice was lower than that in the livers of WT and LXRβ-/- mice. Moreover, induction of hepatic 17β-HSD13 expression by T0901317 was almost completely abolished in SREBP-1c-/- mice. Bioinformatics analysis revealed a consensus sterol regulatory element (SRE)-binding site in the promoter region of the 17β-HSD13 gene. A 17β-HSD13 gene promoter-driven luciferase reporter and ChIP assays further confirmed that 17β-HSD13 gene was under direct control of SREBP-1c. Collectively, these findings demonstrate that LXRα activation induces 17β-HSD13 expression in a SREBP-1c-dependent manner. 17β-HSD13 may be involved in the development of LXRα-mediated fatty liver.

    March 07, 2017   doi: 10.1152/ajpendo.00310.2016   open full text
  • Insulin Sensitizer Prevents and Ameliorates Experimental Type-1 Diabetes.
    Valitsky, M., Hoffman, A., Unterman, T., Bar-Tana, J.
    AJP Endocrinology and Metabolism. March 07, 2017

    Insulin-dependent type-1 diabetes (T1D) is driven by autoimmune beta-cell failure, whereas systemic resistance to insulin is considered the hallmark of insulin-independent type-2 diabetes (T2D). In contrast to this canonical dichotomy, insulin resistance appears to precede the overt diabetic stage of T1D and to predict its progression, implying that insulin sensitizers may change the course of T1D. However, previous attempts to ameliorate T1D in animal models or patients by insulin sensitizers have largely failed. Sensitization to insulin by MEthyl-substituted long-chain DICArboxylic acid (MEDICA) analogs in T2D animal models surpasses that of current insulin sensitizers, thus prompting our interest in probing MEDICA in the T1D context. MEDICA efficacy in modulating the course of T1D was verified in streptozotocin (STZ) diabetic rats and autoimmune non-obese diabetic (NOD) mice. MEDICA treatment normalizes overt diabetes in STZ diabetic rats when added-on to sub-therapeutic insulin, and prevents / delays autoimmune T1D in NOD mice. MEDICA treatment does not improve beta-cell insulin content or insulitis score, but its efficacy is accounted for by pronounced total body sensitization to insulin. In conclusion, potent insulin sensitizers may counteract genetic predisposition to autoimmune T1D, and amplify sub-therapeutic insulin into an effective therapeutic measure for the treatment of overt T1D.

    March 07, 2017   doi: 10.1152/ajpendo.00329.2016   open full text
  • A Four Compartments Compartmental Model to Assess Net Whole Body Protein Breakdown Using a Pulse of Phenylalanine and Tyrosine Stable Isotopes in Humans.
    Mason, A., Engelen, M. P., Ivanov, I., Toffolo, G. M., Deutz, N. E. P.
    AJP Endocrinology and Metabolism. March 07, 2017

    The stable isotopes of Phenylalanine (Phe) and Tyrosine (Tyr) are often used to study whole body protein metabolism in humans. Non-compartmental approaches give limited physiological insight in the compartmental characteristics. We therefore developed a mathematical model of Phe/Tyr metabolism to describe protein fluxes using stable tracer dynamic data in plasma following iv bolus of L-[ring-13C6]-Phe and L-[ring-2H4]-Tyr in healthy subjects. The model consists of four compartments describing Phe/Tyr kinetics. Since the model is a priori non-identifiable, it is quantified in terms of two uniquely identifiable submodels representing two limit case scenarios, based on known physiology. The two submodels, identified by using the software SAAM II, fit well the experimental data of all individuals and provide an unbiased overview of the metabolic pathway in terms of intervals of validity of the non-uniquely identifiable variables. The model provides estimates of the flux from Phe to Tyr (4.1±1.0 µmol•kg ffm-1•h-1, mean±SEM) and intervals of validity of the flux and pool estimates. Our preferred submodel yielded protein breakdown flux (50.5 ± 5.2µmol•kg ffm-1•h-1), net protein breakdown (4.1±1.0µmol•kg ffm-1•h-1), Tyr from Phe hydroxylation (~12%), hydroxylated Phe (~8%) and flux ratio of Tyr/Phe arising from protein catabolism (0.68), consistent with available literature. The other submodel suggest that the assumptions made by non-compartmental analysis are consistently underestimated. Our accurate and detailed model for estimating Phe/Tyr metabolic pathways in humans might be essential in applications in a variety of scenarios describing whole body protein synthesis and breakdown in health and disease.

    March 07, 2017   doi: 10.1152/ajpendo.00362.2016   open full text
  • Skeletal Muscle PGC-1{beta} Signaling is Sufficient to Drive an Endurance Exercise Phenotype and to Counteract Components of Detraining in Mice.
    Lee, S., Leone, T. C., Rogosa, L., Rumsey, J., Ayala, J., Coen, P. M., Fitts, R. H., Vega, R. B., Kelly, D. P.
    AJP Endocrinology and Metabolism. March 07, 2017

    PPARgamma coactivator-1 (PGC-1) α; ;and β serve as master transcriptional regulators of muscle mitochondrial functional capacity and are capable of enhancing muscle endurance when overexpressed in mice. We sought to determine whether muscle-specific transgenic overexpression of PGC-1β affects the detraining response following endurance training. First, we established and validated a mouse exercise training/detraining protocol. Secondly, we found that overexpression of PGC-1β in skeletal muscle of sedentary mice fully recapitulated the training response using multiple physiological and gene expression endpoints. Lastly, overexpression of PGC-1β during the detraining period resulted in a partial prevention of the detraining response. Specifically, an increase in VO2max was maintained in trained mice with muscle overexpression of PGC-1β 6 weeks after cessation of training. However, other detraining responses, including changes in running performance and in situ 1/2 relaxation time (a measure of contractility) were not affected by overexpression of PGC-1β. We conclude that while activation of muscle PGC-1β is sufficient to drive the complete endurance phenotype in sedentary mice, it only partially prevents the detraining response following exercise training suggesting that the process of endurance detraining involves mechanisms beyond the reversal of muscle autonomous mechanisms involved in endurance fitness. In addition, the protocol described here should be useful for assessing early-stage proof-of-concept interventions in pre-clinical models of muscle disuse atrophy.

    March 07, 2017   doi: 10.1152/ajpendo.00380.2016   open full text
  • Alpha-linolenic acid supplementation and exercise training reveal independent and additive responses on hepatic lipid accumulation in obese rats.
    Miotto, P. M., Horbatuk, M., Proudfoot, R., Matravadia, S., Bakovic, M., Chabowski, A., Holloway, G. P.
    AJP Endocrinology and Metabolism. March 07, 2017

    Alpha-linolenic acid (ALA) supplementation or exercise training can independently prevent hepatic lipid accumulation and reduced insulin signaling, however, this may occur through different mechanisms-of-action. In the current study, obese Zucker rats displayed decreased phospholipid (PL) content in association with hepatic lipid abundance, and therefore, we examined whether ALA and exercise training would prevent these abnormalities differently to reveal additive effects on the liver. To achieve this aim, obese Zucker rats were fed control diet alone or supplemented with ALA, and were sedentary or exercise trained for 4 weeks (C-Sed, ALA-Sed, C-Ex, ALA-Ex). ALA-Sed rats had increased microsomal-triglyceride transfer protein (MTTP), a protein required for lipoprotein assembly/secretion, as well as modestly increased phospholipid content in the absence of improvements in mitochondrial content, lipid accumulation, or insulin sensitivity. In contrast, C-Ex rats had increased mitochondrial content and insulin sensitivity, however, this corresponded with minimal improvements in PL content and hepatic lipid accumulation. Importantly, ALA-Ex rats demonstrated additive improvements in PL content and hepatic steatosis, which corresponded with increased mitochondrial content, MTTP and apolipoprotein B100 content, greater serum TAG, and insulin sensitivity. Overall, these data demonstrate additive effects of ALA and exercise training on hepatic lipid accumulation, as exercise-training preferentially increased mitochondrial content, while ALA promoted an environment conducive for lipid secretion. These data highlight the potential for combination therapy to mitigate liver disease progression.

    March 07, 2017   doi: 10.1152/ajpendo.00438.2016   open full text
  • Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiquitin ligases Murf-1 and Atrogin-1 in Cushing's syndrome.
    Kang, S.-H., Lee, H.-A., Kim, M., Lee, E., Sohn, U. D., Kim, I.
    AJP Endocrinology and Metabolism. February 28, 2017

    Cushing's syndrome is caused by overproduction of the adrenocorticotropic hormone (ACTH), which stimulates the adrenal grand to make cortisol. Skeletal muscle wasting occurs in pathophysiological response to Cushing's syndrome. The forkhead box (FOX) protein family has been implicated as a key regulator of muscle loss under conditions such as diabetes and sepsis. However, the mechanistic role of the FOXO family in ACTH-induced muscle atrophy is not understood. We hypothesized that FOXO3a plays a role in muscle atrophy through expression of the E3 ubiquitin ligases, muscle RING finger protein-1 (MuRF-1) and Atrogin-1 in Cushing's syndrome. For establishment of a Cushing's syndrome animal model, Sprague-Dawley rats were implanted with osmotic mini-pumps containing ACTH (40 ng/kg/day). ACTH infusion significantly reduced muscle weight. In ACTH-infused rats, MuRF-1, Atrogin-1, and FOXO3a were upregulated and FOXO3a promoter was targeted by glucocorticoid receptor (GR). Transcriptional activity and expression of FOXO3a was significantly decreased by GR antagonist RU486. Treatment with RU486 reduced MuRF-1 and Atrogin-1 expression in accordance with reduced enrichment of FOXO3a and Pol II on its promoters. Knockdown of FOXO3a prevented Dex-induced MuRF-1 and Atrogin-1 expression. These results indicate that FOXO3a plays a role in muscle atrophy through expression of MuRF-1 and Atrogin-1 in Cushing's syndrome.

    February 28, 2017   doi: 10.1152/ajpendo.00389.2016   open full text
  • Insulin resistance and normal thyroid hormone levels: prospective study and metabolomic analysis.
    Ferrannini, E., Iervasi, G., Cobb, J., Ndreu, R., Nannipieri, M.
    AJP Endocrinology and Metabolism. February 28, 2017

    While hyper/hypothyroidism causes dysglycemia, the relationship between thyroid hormone levels within the normal range and insulin resistance (IR) is unclear. In 940 participants with strictly normal serum concentrations of free triiodothyronine (fT3), free thyroxine (fT4), and thyroid-stimulating hormone (TSH)) followed up for 3 years, we measured insulin sensitivity (by the insulin clamp technique) and a panel of 35 circulating metabolites. At baseline, across quartiles of increasing fT3 levels (or fT3/fT4 ratio) there emerged most features of IR (male sex, higher BMI, waist circumference, heart rate, blood pressure, fatty liver index, free fatty acids, and triglycerides levels, reduced insulin-mediated glucose disposal and ß-cell glucose sensitivity). In multiadjusted analyses, fT3 was reciprocally related to insulin sensitivity and, in a subset of 303 subjects, directly related to endogenous glucose production. In multiple regression models adjusting for sex, age, BMI and baseline value of insulin sensitivity, higher baseline fT3 levels were significant predictors of the decreases in insulin sensitivity. Moreover, baseline fT3 predicted follow-up increases in glycemia independently of sex, age, BMI, insulin sensitivity, ß-cell glucose sensitivity and baseline glycemia. Serum tyrosine levels were higher in IR and were directly associated with fT3; higher α–hydroxybutyrate levels signaled enhanced oxidative stress impairing tyrosine degradation. In 25 morbidly obese patients, surgery-induced weight loss improved IR and consensually lowered fT3. High-normal fT3 levels are associated with IR both cross-sectionally and longitudinally, and predict deterioration of glucose tolerance. This association is supported by a metabolite pattern that points at increased oxidative stress as part of the IR syndrome.

    February 28, 2017   doi: 10.1152/ajpendo.00464.2016   open full text
  • CTRP7 deletion attenuates obesity-linked glucose intolerance, adipose tissue inflammation, and hepatic stress.
    Petersen, P. S., Lei, X., Wolf, R. M., Rodriguez, S., Tan, S. Y., Little, H. C., Schweitzer, M. A., Magnuson, T. H., Steele, K. E., Wong, G. W.
    AJP Endocrinology and Metabolism. February 21, 2017

    Chronic low-grade inflammation and cellular stress are important contributors to obesity-linked metabolic dysfunction. Here, we uncover an immune-metabolic role for C1q/TNF-related protein 7 (CTRP7), a secretory protein of the C1q family with previously unknown function. In obese humans, circulating CTRP7 levels were markedly elevated and positively correlated with BMI, glucose, insulin, insulin resistance index, hemoglobin A1c, and triglyceride levels. Expression of CTRP7 in liver was also significantly upregulated in obese humans and positively correlated with gluconeogenic genes. In mice, Ctrp7 expression was differentially modulated in various tissues by fasting and re-feeding and by diet-induced obesity. A genetic loss-of-function mouse model was used to determine the requirement of CTRP7 for metabolic homeostasis. When fed a control low-fat diet, male or female mice lacking CTRP7 were indistinguishable from wild-type littermates. In obese male mice consuming a high-fat diet, however, CTRP7 deficiency attenuated insulin resistance and enhanced glucose tolerance, effects that were independent of body weight, metabolic rate, and physical activity level. Improved glucose metabolism in CTRP7-deficient mice was associated with reduced adipose tissue inflammation as well as decreased liver fibrosis and cellular oxidative and endoplasmic reticulum stress. These results provide a link between elevated CTRP7 levels and impaired glucose metabolism frequently associated with obesity. Inhibiting CTRP7 action may confer beneficial metabolic outcomes in the setting of obesity and diabetes.

    February 21, 2017   doi: 10.1152/ajpendo.00344.2016   open full text
  • Muscle PGC-1{alpha} is required for long term systemic and local adaptations to a ketogenic diet in mice.
    Schnyder, S., Svensson, K., Cardel, B., Handschin, C.
    AJP Endocrinology and Metabolism. February 21, 2017

    Low carbohydrate/high-fat (LCHF) diets are increasingly popular dietary interventions for body weight control and as treatment for different pathological conditions. However, the mechanisms of action are still poorly understood, in particular in long-term administration. Besides liver, brain and heart, skeletal muscle is one of the major organs involved in the regulation of physiological and pathophysiological ketosis. We now assessed the role of the peroxisome proliferator-activated receptor coactivator 1α (PGC-1α) in skeletal muscle of male wild type control (CTRL) and PGC-1α muscle-specific knockout (PGC-1α mKO) mice upon 12 weeks of LCHF diet feeding. Interestingly, LCHF diet administration increased oxygen consumption in a muscle PGC-1α-dependent manner concomitant with a blunted transcriptional induction of genes involved in fatty acid oxidation and impairment in exercise performance. These data reveal a new role for muscle PGC-1α in regulating the physiological adaptation to long-term LCHF diet administration.

    February 21, 2017   doi: 10.1152/ajpendo.00361.2016   open full text
  • The Loss Of Macrophage Fatty Acid Oxidation Does Not Potentiate Systemic Metabolic Dysfunction.
    Gonzalez-Hurtado, E., Lee, J., Choi, J., Selen Alpergin, E. S., Collins, S. L., Horton, M. R., Wolfgang, M. J.
    AJP Endocrinology and Metabolism. February 21, 2017

    Fatty acid oxidation in macrophages has been suggested to play a causative role in high-fat diet-induced metabolic dysfunction, particularly in the etiology of adipose driven insulin resistance. To understand the contribution of macrophage fatty acid oxidation directly to metabolic dysfunction in high-fat diet-induced obesity, we generated mice with a myeloid-specific knockout of carnitine palmitoyltransferase 2 (CPT2 M-KO), an obligate step in mitochondrial long-chain fatty acid oxidation. While fatty acid oxidation was clearly induced upon IL-4 stimulation, fatty acid oxidation deficient CPT2 M-KO bone marrow derived macrophages (BMDM) displayed canonical markers of M2 polarization following IL-4 stimulation in vitro. In addition, loss of macrophage fatty acid oxidation in vivo did not alter the progression of high-fat diet induced obesity, inflammation, macrophage polarization, oxidative stress, or glucose intolerance. These data suggest that although alternatively activated macrophages up-regulate fatty acid oxidation, fatty acid oxidation is dispensable for macrophage polarization and high-fat diet-induced metabolic dysfunction. Macrophage fatty acid oxidation likely plays a correlative rather than causative role in systemic metabolic dysfunction.

    February 21, 2017   doi: 10.1152/ajpendo.00408.2016   open full text
  • The {beta}3-adrenergic receptor is dispensable for browning of adipose tissues.
    de Jong, J. M. A., Wouters, R. T. F., Boulet, N., Cannon, B., Nedergaard, J., Petrovic, N.
    AJP Endocrinology and Metabolism. February 21, 2017

    Brown and brite/beige adipocytes are attractive therapeutic targets to treat metabolic diseases. To maximally utilize their functional potential, further understanding is required about their identities and their functional differences. Recent studies with β3-adrenergic receptor knockout mice reported that brite/beige adipocytes, but not classical brown adipocytes, require the β3-adrenergic receptor for cold-induced transcriptional activation of thermogenic genes. We aimed to further characterize this requirement of the β3-adrenergic receptor as a functional distinction between classical brown and brite/beige adipocytes. However, when comparing wild-type and β3-adrenergic receptor knockout mice, we observed no differences in cold-induced thermogenic gene expression (Ucp1, Pgc1a, Dio2 and Cidea) in brown or white (brite/beige) adipose tissues. Irrespective of the duration of the cold exposure or the sex of the mice, we observed no effect of the absence of the β3-adrenergic receptor. Experiments with the β3-adrenergic receptor agonist CL-316,243 verified the functional absence of β3-adrenergic signaling in these knockout mice. The β3-adrenergic receptor knockout model in the present study was maintained on a FVB/N background, whereas earlier reports used C57BL/6 and 129Sv mice. Thus, our data imply background-dependent differences in adrenergic signaling mechanisms in response to cold exposure. Nonetheless, the present data indicate that the β3-adrenergic receptor is dispensable for cold-induced transcriptional activation in both classical brown and, as opposed to earlier studies, brite/beige cells. This should be taken into account in the increasing number of studies on the induction of browning and their extrapolation to human physiology.

    February 21, 2017   doi: 10.1152/ajpendo.00437.2016   open full text
  • A Model Of Chronic Diabetic Polyneuropathy: Benefits From Intranasal Insulin Are Modified By Sex And Rage Deletion.
    de la Hoz, C. L., Cheng, C., Fernyhough, P., Zochodne, D. W.
    AJP Endocrinology and Metabolism. February 21, 2017

    Human diabetic polyneuropathy (DPN) is a progressive complication of chronic diabetes mellitus. Preliminary evidence has suggested that intranasal insulin, in doses insufficient to alter hyperglycemia, suppresses the development of DPN. In this work we confirm this finding, but demonstrate that its impact is modified by sex and deletion of RAGE, the receptor for advanced glycosylation endproducts. We serially evaluated experimental DPN in male and female wild type mice and male RAGE null (RN) mice, each with nondiabetic controls, during 16 weeks of diabetes, the final 8 weeks including groups given intranasal insulin. Age matched nondiabetic female mice had higher motor and sensory conduction velocities than their male counterparts and had lesser conduction slowing from chronic diabetes. Intranasal insulin improved slowing in both genders. In male RN mice, there was lesser conduction slowing with chronic diabetes and intranasal insulin provided limited benefits. Rotarod testing, and hindpaw grip power offered less consistent impacts . Mechanical sensitivity and thermal sensitivity were respectively but disparately changed and improved with insulin in wild type female and male mice but not RN male mice. These studies confirm that intranasal insulin improves indices of experimental DPN but indicates that females with DPN may differ in their underlying phenotype. RN mice had partial but incomplete protection from underlying DPN and lesser impacts from insulin. We also identify an important role for sex in the development of DPN and report evidence that insulin and AGE-RAGE pathways in its pathogenesis may overlap.

    February 21, 2017   doi: 10.1152/ajpendo.00444.2016   open full text
    Chan, K. L., Boroumand, P., Milanski, M., Pillon, N. J., Bilan, P. J., Klip, A.
    AJP Endocrinology and Metabolism. February 14, 2017

    Over the past years we have embarked in a systematic analysis of the effect of obesity or fatty acids on circulating monocytes, microvascular endothelial cells, macrophages and skeletal muscle cells. Using cell culture strategies, we have deconstructed complex physiological systems, then reconstructed 'partial equations' to better understand cell-to-cell communication. Through these approaches we identified that in high saturated fat environments, cell-autonomous pro-inflammatory pathways are activated in monocytes and endothelial cells, promoting monocyte adhesion and transmigration. We think of this as a paradigm of the conditions promoting immune cell infiltration into tissues during obesity. In concert, it is possible that muscle and adipose tissue secrete immune cell chemoattractants, and indeed our tissue culture reconstructions reveal that myotubes treated with the saturated fatty acid palmitate, but not the unsaturated fatty acid palmitoleate, release nucleotides that attract monocytes, and other compounds that promote pro-inflammatory 'M1-like' polarization in macrophages. In addition, palmitate directly triggers an M1-like macrophage phenotype, and secretions from thus activated macrophages confer insulin resistance to target muscle cells. Together, these studies suggest that in pathophysiological conditions of excess fat, the muscle, endothelial and immune cells engage in a synergistic crosstalk that exacerbates tissue inflammation, leukocyte infiltration, polarization, and consequent insulin resistance.

    February 14, 2017   doi: 10.1152/ajpendo.00039.2017   open full text
  • Dietary nitrite reverses features of postmenopausal metabolic syndrome induced by high fat diet and ovariectomy in mice.
    Ohtake, K., Ehara, N., Chiba, H., Nakano, G., Sonoda, K., Ito, J., Uchida, H., Kobayashi, J.
    AJP Endocrinology and Metabolism. February 14, 2017

    Menopausal women are at greater risk of developing metabolic syndrome with reduced endothelial nitric oxide synthase (eNOS) activity. Hormone replacement therapy increases eNOS activity and normalizes some characteristics of metabolic syndrome. We hypothesized that nitric oxide (NO) supplementation should have a therapeutic effect in this syndrome. We examined the effect of dietary nitrite on mice model with postmenopausal metabolic syndrome induced by ovariectomy (OVX) with high fat diet (HF). C57BL/6 female mice were divided into five groups, sham+normal fat diet (NF), sham+ HF, OVX+HF without or with sodium nitrite (50mg and 150mg/L) in drinking water. Daily food intake and weekly body weight were monitored for 18 weeks. OVX and HF significantly reduced plasma levels of nitrate/nitrite (NOx), and developed obesity with visceral hypertrophic adipocytes, and increased transcriptional levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrotizing factor-α (TNF-α) and interleukin-6 (IL-6) in visceral fat tissues. The proinflammatory state in the adipocytes provoked severe hepatosteatosis and insulin resistance in OVX+HF group compared with sham+NF group. However, dietary nitrite significantly suppressed adipocyte hypertrophy and transcriptions of proinflammatory cytokines in visceral fat in a dose dependent manner. The improvement of visceral inflammatory state consequently reversed the hepatosteatosis and insulin resistance observed in OVX+HF mice. These results suggest that endogenous NO defect might underlie postmenopausal metabolic syndrome, and dietary nitrite provides an alternative source of NO, and subsequently compensating for metabolic impairments of this syndrome.

    February 14, 2017   doi: 10.1152/ajpendo.00360.2016   open full text
  • S100B impairs glycolysis via enhanced poly(ADP-ribosyl)ation of glyceraldehyde 3-phosphate dehydrogenase in rodent muscle cells.
    Hosokawa, K., Hamada, Y., Fujiya, A., Murase, M., Maekawa, R., Niwa, Y., Izumoto, T., Seino, Y., Tsunekawa, S., Arima, H.
    AJP Endocrinology and Metabolism. February 07, 2017

    S100 calcium-binding protein B (S100B), a multifunctional macromolecule mainly expressed in nerve tissues and adipocytes, has been suggested to contribute to the pathogenesis of obesity. To clarify the role of S100B in insulin action and glucose metabolism in peripheral tissues, we investigated the effect of S100B on glycolysis in myoblast and myotube cells. Rat myoblast L6 cells were treated with recombinant mouse S100B to examine glucose consumption, lactate production, glycogen accumulation, glycolytic metabolites and enzyme activity, insulin signaling, and poly(ADP-ribosyl)ation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Glycolytic metabolites were investigated by enzyme assays or metabolome analysis, and insulin signaling was assessed by western blot analysis. Enzyme activity and poly(ADP-ribosyl)ation of GAPDH was evaluated by an enzyme assay and immunoprecipitation followed by dot blot with an anti-poly(ADP-ribose) antibody, respectively. S100B significantly decreased glucose consumption, glucose analog uptake, and lactate production in L6 cells, in either the presence or absence of insulin. In contrast, S100B had no effect on glycogen accumulation and insulin signaling. Metabolome analysis revealed that S100B increased the concentration of glycolytic intermediates upstream of GAPDH. S100B impaired GAPDH activity and increased poly(ADP-ribosyl)ated GAPDH proteins. The effects of S100B on glucose metabolism were mostly canceled by a poly(ADP-ribose) polymerase (PARP) inhibitor. Similar results were obtained in C2C12 myotube cells. We conclude that S100B as a humoral factor may impair glycolysis in muscle cells independently of insulin action, and the effect may be attributed to the inhibition of GAPDH activity from enhanced poly(ADP-ribosyl)ation of the enzyme.

    February 07, 2017   doi: 10.1152/ajpendo.00328.2016   open full text
  • Suprachiasmatic Vasopressin to Paraventricular Oxytocin Neurocircuit in the Hypothalamus Relays Light Reception to Inhibition of Feeding Behavior.
    Santoso, P., Nakata, M., Ueta, Y., Yada, T.
    AJP Endocrinology and Metabolism. February 07, 2017

    Light syncronizes body's circadian rhythms by modulating the master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. In modern life, the extended and/or irregular light exposure impairs circadian rhythms and consequently promotes feeding and metabolic disorders. However, the neuronal pathway through which light is coupled to feeding behavior is less elucidated. The present study employed the light exposure during dark phase of the day in rats and observed its effect on neuronal activity and feeding behavior. Light exposure acutely suppressed food intake and elevated c-Fos expression in the arginine vasopressin (AVP) neurons of SCN and the oxytocin (Oxt) neurons of paraventricular nucleus (PVN) in the hypothalamus. The light-induced suppression of food intake was abolished by blockade of the Oxt receptor in the brain. Retrograde tracer analysis demonstrated the projection of SCN AVP neurons to PVN. Furthermore, intracerebroventricular (icv) injection of AVP suppressed food intake and increased c-Fos in PVN Oxt neurons. Intra-PVN injection of AVP exerted a stronger anorexigenic effect than icv injection. AVP also induced intracellular Ca2+ signaling and increased firing frequency in Oxt neurons in PVN slices. These results reveal the novel neurocircuit from SCN AVP to PVN Oxt that relays light reception to inhibition of feeding behavior. This light-induced neurocircuit may serve as a pathway for forming the circadian feeding rhythm, and linking irregular light exposure to arrhythmic feeding and consequently obesity and metabolic diseases.

    February 07, 2017   doi: 10.1152/ajpendo.00338.2016   open full text
  • Cardiovascular benefits associated with higher dietary K+ versus lower dietary Na+: Evidence from population and mechanistic studies.
    McDonough, A. A., Veiras, L. C., Guevara, C. A., Ralph, D. L.
    AJP Endocrinology and Metabolism. February 07, 2017

    The WHO ranks hypertension the leading global risk factor for disease, specifically, cardiovascular disease. Blood pressure is higher in westernized populations consuming sodium-rich processed foods compared to isolated societies consuming potassium-rich natural foods. Evidence suggests that lowering dietary Na+ is particularly beneficial in hypertensives who consume a high Na+ diet. Nonetheless, numerous population studies demonstrate a relationship between higher dietary K+, estimated from urinary excretion or dietary recall, and lower blood pressure regardless of sodium intake. Interventional studies with potassium supplementation suggest it provides a direct benefit; K+ may also be a marker for other beneficial components of a "natural" diet. Recent studies in rodent models indicate mechanisms for the potassium benefit: the distal tubule Na+- Cl- cotransporter (NCC) controls Na+ delivery downstream to the collecting duct where Na+ reabsorbed by epithelial Na+ channels (ENaC) drives K+ secretion and excretion through K+ channels in the same region. High dietary K+ provokes a decrease in the NCC activity to drive more K+ secretion (and Na+ excretion, analogous to the actions of a thiazide diuretic) whether Na+ intake is high or low; low dietary K+ provokes an increase in NCC activity and Na+ retention, also independent of dietary Na+. Taken together, the findings suggest that public health efforts directed towards increasing consumption of natural potassium rich foods would reduce blood pressure and, thus, cardiovascular and kidney disease.

    February 07, 2017   doi: 10.1152/ajpendo.00453.2016   open full text
  • Muscle interleukin 6 and fasting-induced PDH regulation in mouse skeletal muscle.
    Gudiksen, A., Bertholdt, L., Vingborg, M. B., Hansen, H. W., Ringholm, S., Pilegaard, H.
    AJP Endocrinology and Metabolism. February 07, 2017

    Fasting prompts a metabolic shift in substrate utilization from carbohydrate to predominant fat oxidation in skeletal muscle and pyruvate dehydrogenase (PDH) is seen as a controlling link between the competitive oxidation of carbohydrate and fat during metabolic challenges like fasting. Interleukin (IL)-6 has been proposed to be released from muscle with concomitant effects on both glucose and fat utilization. The aim was to test the hypothesis that IL-6 has a regulatory impact on fasting-induced suppression of skeletal muscle PDH. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (Control) were either fed or fasted for 6 or 18 hours. Lack of muscle IL-6 elevated the respiratory exchange ratio (RER) in the fed and early fasting state, but not with prolonged fasting. PDHa activity was higher in fed and fasted IL-6 MKO than Control mice, while lack of muscle IL-6 did not prevent down-regulation of PDHa activity in skeletal muscle or changes in plasma and muscle substrate levels in response to 18h of fasting. Phosphorylation of 3 out of 4 sites on PDH-E1α increased with 18h of fasting, but was lower in IL-6 MKO mice than Control. In addition, both PDK4 mRNA and protein increased with 6h and 18h of fasting in both genotypes, but PDK4 protein was lower in IL-6 MKO than Control. In conclusion, muscle IL-6 seems to regulate resting substrate utilization potentially through effects on skeletal muscle PDH regulation, but is not required for maintaining metabolic flexibility in response to fasting.

    February 07, 2017   doi: 10.1152/ajpendo.00291.2016   open full text
  • "Nutraceuticals" in relation to human skeletal muscle and exercise.
    Deane, C. S., Wilkinson, D. J., Phillips, B. E., Smith, K., Etheridge, T., Atherton, P. J.
    AJP Endocrinology and Metabolism. January 31, 2017

    Skeletal muscles have a fundamental role in locomotion and whole body metabolism, with muscle mass and quality being linked to improved health and even lifespan. Optimising nutrition in combination with exercise is considered an established, effective ergogenic practice for athletic performance. Importantly, exercise and nutritional approaches also remain arguably the most effective countermeasure for muscle dysfunction associated with ageing and numerous clinical conditions e.g. cancer cachexia, COPD and organ failure, via engendering favourable adaptations such as increased muscle mass and oxidative capacity. Therefore, it is important to consider the effects of established and novel effectors of muscle mass, function and metabolism in relation to nutrition and exercise. To address this gap, in this review we detail existing evidence surrounding the efficacy of a non-exhaustive list of macronutrient, micronutrient and "nutraceutical" compounds alone and in combination with exercise in relation to skeletal muscle mass, (protein and fuel) metabolism and exercise performance (i.e. strength and endurance capacity). It is long established that macronutrients have specific roles and impacts upon protein metabolism and exercise performance i.e. protein positively influences muscle muscle mass and protein metabolism, whilst carbohydrate and fat intakes can influence fuel metabolism and exercise performance. Regarding novel nutraceuticals, we show the following ones in particular may have effects in relation to: 1) muscle mass/protein metabolism: leucine, hydroxyl b-methylbutyrate, creatine, vitamin-D, ursolic acid and phosphatidic acid, and 2) exercise performance: (i.e. strength or endurance capacity); hydroxyl -methylbutyrate, carnitine, creatine, nitrates and b-alanine.

    January 31, 2017   doi: 10.1152/ajpendo.00230.2016   open full text
  • Timing of Meals: When is as Critical as What and How Much.
    Jiang, P., Turek, F. W.
    AJP Endocrinology and Metabolism. January 31, 2017

    Over the past decade, a large body of literature has demonstrated that disruptions of the endogenous circadian clock, either environmental or genetic, lead to metabolic dysfunctions associated with obesity, diabetes, and other metabolic disorders. The phrase, "It is not only what you eat and how much you eat, but also when you eat" sends a simple message about circadian timing and body weight regulation. Getting this message out to clinicians and patients, while at the same time the elucidation of neuroendocrine, molecular, and genetic mechanisms underlying this phrase makes it very likely that the circadian impact on lifestyle and clinical practice could be soon adopted for improving overall human health. In this review, we discuss findings from animal models as well as epidemiological and clinical studies in humans, which collectively promote the awareness of the role of circadian clock in metabolic functions and dysfunctions.

    January 31, 2017   doi: 10.1152/ajpendo.00295.2016   open full text
  • Chronic hyperinsulinemia contributes to insulin resistance under dietary restriction in association with altered lipid metabolism in Zucker diabetic fatty rats.
    Morita, I., Tanimoto, K., Akiyama, N., Naya, N., Fujieda, K., Iwasaki, T., Yukioka, H.
    AJP Endocrinology and Metabolism. January 31, 2017

    Hyperinsulinemia is widely thought to be a compensatory response to insulin resistance, whereas its potentially causal role in the progression of insulin resistance remains to be established. Here, we aimed to examine whether hyperinsulinemia could affect the progression of insulin resistance in Zucker fatty diabetic (ZDF) rats. Male ZDF rats, at 8 weeks of age, were fed a diet ad libitum (AL) or DR of either 15% or 30% from AL feeding over 6 weeks. Insulin sensitivity was determined by hyperinsulinemic-euglycemic clamp. ZDF rats in AL group progressively developed hyperglycemia and hyperinsulinemia by 10 weeks of age, and then plasma insulin rapidly declined to near normal levels by 12 weeks of age. Compared with AL group, DR groups showed delayed onset of hyperglycemia and persistent hyperinsulinemia, leading to weight gain and raised plasma triglyceride and free fatty acid by 14 weeks of age. Notably, insulin sensitivity was significantly reduced in DR group rather than AL group, and was inversely correlated with plasma levels of insulin and triglyceride, but not glucose. Moreover, enhanced lipid deposition and up-regulation of genes involved in lipogenesis were detected in liver, skeletal muscle and adipose tissues of DR group rather than AL group. Alternatively, continuous hyperinsulinemia induced by insulin pellet implantation produced a decrease in insulin sensitivity in ZDF rats. These results suggest that chronic hyperinsulinemia may lead to the progression of insulin resistance under DR conditions in association with altered lipid metabolism in peripheral tissues in ZDF rats.

    January 31, 2017   doi: 10.1152/ajpendo.00342.2016   open full text
  • Phenotyping of Adipose, Liver, and Skeletal Muscle Insulin Resistance and Response to Pioglitazone in Spontaneously Obese Rhesus Monkeys.
    Shang, J., Previs, S. F., Conarello, S., Chng, K., Zhu, Y., Souza, S. C., Staup, M., Chen, Y., Xie, D., Zycband, E., Schlessinger, K., Plamadeala Johnson, V., Arreaza, G., Liu, F., Levitan, D., Wang, L., van Heek, M., Erion, M., Wang, Y., Kelley, D. E.
    AJP Endocrinology and Metabolism. January 31, 2017

    Insulin resistance and diabetes can develop spontaneously with obesity and aging in rhesus monkeys, highly similar to the natural history of obesity, insulin resistance and progression to type 2 diabetes in humans. The current studies in obese rhesus were undertaken to assess hepatic and adipose contributions to systemic insulin resistance, currently a gap in our knowledge, and to benchmark the responses to pioglitazone (PIO). A 2-step hyperinsulinemic-euglycemic clamp, with tracer-based glucose flux estimates, was used to measure insulin resistance and in an intervention study was repeated following six weeks of PIO treatment (3 mg/kg). Compared to lean healthy rhesus, obese rhesus has a 60% reduction of glucose utilization during a high insulin infusion and markedly impaired suppression of lipolysis evident at both low and high insulin infusion. However, obese dysmetabolic rhesus manifests only mild hepatic insulin resistance. Six-week PIO treatment significantly improved skeletal muscle and adipose insulin resistance (by ~50%). These studies strengthen the concept that insulin resistance in obese rhesus closely resembles human insulin resistance, and indicate the value of obese rhesus for appraising new insulin-sensitizing therapeutics.

    January 31, 2017   doi: 10.1152/ajpendo.00398.2016   open full text
  • Exogenous H2S regulates endoplasmic reticulum-mitochondria cross-talk to inhibit apoptotic pathways in STZ-induced type I diabetes.
    Yang, F., Yu, X., Li, T., Wu, J., Zhao, Y., Liu, J., Sun, A., Dong, S., Wu, J., Zhong, X., Xu, C., Lu, F., Zhang, W.
    AJP Endocrinology and Metabolism. January 31, 2017

    Background: The upregulation of reactive oxygen species (ROS) is a primary cause of cardiomyocyte apoptosis in Diabetes cardiomyopathy (DCM). Mitofusin-2 (Mfn-2) is a key protein that bridges the mitochondria and endoplasmic reticulum(ER). Hydrogen sulfide (H2S)-mediated cardioprotection is related to antioxidant effects. The present study demonstrated that H2S inhibited the interaction between the ER and mitochondrial apoptotic pathway. Methods: This study investigated cardiac function, ultrastructural changes in the ER and mitochondria, apoptotic rate using TUNEL and the expression of ER stress-associated proteins and mitochondrial apoptotic proteins in cardiac tissues in STZ-induced type I diabetic rats treated with or without NaHS (donor of H2S). Mitochondria of cardiac tissues were isolated, and MPTP opening and cytochrome C (cyt C) and Mfn-2 expression were also detected. Results: Our data showed that hyperglycemia decreased the cardiac function by Ultrasound Cardiogram, and the administration of exogenous H2S ameliorated these changes. We demonstrated that the expression of ER stress sensors and apoptotic rates were elevated in cardiac tissue of DCM and cultured H9C2 cells, but the expression of these proteins was reduced following exogenous H2S treatment. The expression of mitochondrial apoptotic proteins, cyt C and mPTP opening were decreased following treatment with exogenous H2S. In our experiment, the expression and immunofluorescence of Mfn-2 were both decreased after transfection with Mfn-2-siRNA. Conclusion: Hyperglycemia stimulated ER interactions and mitochondrial apoptotic pathways, which were inhibited by exogenous H2S treatment through the regulation of Mfn-2 expression.

    January 31, 2017   doi: 10.1152/ajpendo.00196.2016   open full text
  • Mice with hyperbilirubinemia due to Gilbert's Syndrome polymorphism are resistant to hepatic steatosis by decreased serine 73 phosphorylation of PPAR{alpha}.
    Hinds, T. D., Hosick, P. A., Hankins, M. W., Nestor-Kalinoski, A., Stec, D. E.
    AJP Endocrinology and Metabolism. January 17, 2017

    Gilbert's syndrome is derived from a polymorphism (TA repeat) in the hepatic UGT1A1 gene which results in decreased conjugation and increased levels of unconjugated bilirubin. Recently, we have shown that bilirubin binds directly to the fat burning nuclear peroxisome proliferator-activated receptor α (PPARα). Additionally, we have shown that serine 73 phosphorylation (Ser(P)73) of PPARα decreases activity by reducing its protein levels and transcriptional activity. The aim of this study was to determine if humanized mice with the Gilbert's polymorphism (HuUGT*28) have increased PPARα activation and reduced hepatic fat accumulation. To determine if humanized mice with Gilbert's mutation (HuUGT*28) have reduced hepatic lipids we placed them and C57BL/6J control mice on a high fat (60%) diet for 36 weeks. Body weights, fat and lean mass, fasting blood glucose and insulin levels were measured every six weeks throughout the investigation. At the end of the study, hepatic lipid content was measured and PPARα regulated genes as well as immunostaining of Ser(P)73 PPARα from liver sections. The HuUGT*28 mice had increased serum bilirubin, lean body mass, and decreased fat mass, hepatic lipid content, as well as lower serum glucose and insulin levels. Also, the HuUGT*28 mice had reduced Ser(P)73 PPARα immunostaining in livers and increased PPARα transcriptional activity as compared to controls. A chronic, but mild endogenous increase in unconjugated hyperbiliubinemia protects against hepatic steatosis through a reduction in Ser(P)73 PPARα causing an increase in PPARα transcriptional activity and fat burning.

    January 17, 2017   doi: 10.1152/ajpendo.00396.2016   open full text
  • Overexpression of PGC-1{alpha} Increases Peroxisomal and Mitochondrial Fatty Acid Oxidation in Human Primary Myotubes.
    Huang, T.-Y., Zheng, D., Houmard, J. A., Brault, J. J., Hickner, R. C., Cortright, R. N.
    AJP Endocrinology and Metabolism. January 10, 2017

    Peroxisomes are indispensable organelles for lipid metabolism in humans and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI =24.0 ± 0.6 kg/m2, N = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (Peroxins) and genes (PEXs) responsible for proliferation and functions were assessed by western blotting and real-time qRT-PCR respectively. 1-14C palmitic acid and 1-14C lignoceric acid (exclusive peroxisomal specific substrate) were used to assess mitochondrial oxidation of peroxisomal derived metabolites. Following overexpression of PGC-1α, 1) Peroxisomal membrane protein 70kD (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated (P<0.05) 2) PGC-1α, PMP70, key PEXs, and peroxisomal β-oxidation mRNA expression levels were significantly upregulated (P<0.05) and 3) A concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed (P<0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomes and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation as observed in HSkM cells.

    January 10, 2017   doi: 10.1152/ajpendo.00331.2016   open full text
  • Glucose-dependent Insulinotropic Polypeptide promotes lipid deposition in subcutaneous adipocytes in obese, type 2 diabetes patients: a maladaptive response.
    Thondam, S. K., Daousi, C., Wilding, J. P. H., Holst, J. J., Ameen, G. I., Yang, C., Whitmore, C., Mora, S., Cuthbertson, D. J.
    AJP Endocrinology and Metabolism. January 10, 2017

    Glucose-dependent insulinotropic polypeptide (GIP) beyond its insulinotropic effects may regulate post-prandial lipid metabolism. While the insulinotropic action of GIP is known to be impaired in type 2 diabetes mellitus (T2DM), its adipogenic effect is unknown. We hypothesised GIP is anabolic in human subcutaneous adipose tissue (SAT) promoting triacylglycerol (TAG) deposition through re-esterification of non-esterified fatty acids (NEFA) and this effect may differ according to obesity status or glucose tolerance. Methods: 23 subjects, categorised in four groups: normoglycaemic lean (n=6), normoglycaemic obese, (n=6), obese with impaired glucose regulation (IGR) (n=6) and obese, T2DM (n=5) participated in a double-blind, randomised, crossover study involving a hyperglycaemic clamp with a 240 minute GIP infusion (2pmol kg-1min-1) or normal saline. Insulin, NEFA, SAT-TAG content and gene expression of key lipogenic enzymes were determined before and immediately after GIP/saline infusions. Results: GIP lowered NEFA concentrations in obese T2DM group despite diminished insulinotropic activity (mean NEFA AUC0-4hr ± SEM, 41992 ±9843 µmol/L/min vs 71468 ±13605 with placebo, p=0.039; 95% CI 0.31 to 0.95). Additionally, GIP increased SAT-TAG in obese T2DM (1.78 ±0.4 vs 0.86 ±0.1 fold with placebo, p=0.043; 95% CI: 0.1 to 1.8). Such effect with GIP was not observed in other three groups despite greater insulinotropic activity. Reduction in NEFA concentration with GIP correlated with adipose tissue insulin resistance for all subjects (Pearson r=0.56, p=0.005). There were no significant gene expression changes in key SAT lipid metabolism enzymes. Conclusion: GIP appears to promote fat accretion and thus may exacerbate obesity and insulin resistance in T2DM.

    January 10, 2017   doi: 10.1152/ajpendo.00347.2016   open full text
  • Chronic kidney disease and obesity bias surrogate estimates of insulin sensitivity compared to the hyperinsulinemic-euglycemic clamp.
    Ahmad, I., Zelnick, L. R., Robinson, N. R., Hung, A. M., Kestenbaum, B., Utzschneider, K. M., Kahn, S. E., de Boer, I. H.
    AJP Endocrinology and Metabolism. January 10, 2017

    Introduction: Insulin sensitivity can be measured by procedures such as the hyperinsulinemic-euglycemic clamp or using surrogate indices. Chronic kidney disease (CKD) and obesity may differentially affect these measurements because of changes in insulin kinetics and organ-specific effects on insulin sensitivity. Methods: In a cross-sectional study of 59 subjects with non-diabetic CKD (estimated glomerular filtration rate (GFR) <60 mL/min/1.73 m2) and 39 matched healthy controls, we quantified insulin sensitivity by clamp (SIclamp), oral glucose tolerance test, and fasting glucose and insulin. We compared surrogate insulin sensitivity indices to SIclamp using descriptive statistics, graphical analyses, correlation coefficients, and linear regression. Results: Mean age was 62.6 years, 48% of participants were female, and 77% were Caucasian. Insulin sensitivity indices were 8% to 38% lower in participants with versus without CKD and 13% to 59% lower in obese compared to non-obese participants. Correlations of surrogate indices with SIclamp did not significantly differ by CKD or obesity status. Adjusting for SIclamp in addition to demographic factors, Matsuda index was 15% lower in participants with versus without CKD (p=0.09) and 36% lower in participants with versus without obesity (p=0.0001), while 1/HOMA-IR was 23% lower in participants with versus without CKD (p=0.02) and 46% lower in participants with versus without obesity (p<0.0001). Conclusions: CKD and obesity do not significantly alter correlations of surrogate insulin sensitivity indices with SIclamp, but do bias surrogate measurements of insulin sensitivity toward lower values. This bias may be due to differences in insulin kinetics or organ-specific responses to insulin.

    January 10, 2017   doi: 10.1152/ajpendo.00394.2016   open full text
  • Regulation of the Macrophage Oxytocin Receptor in Response to Inflammation.
    Szeto, A., Sun-Suslow, N., Mendez, A. J., Hernandez, R. I., Wagner, K. V., McCabe, P. M.
    AJP Endocrinology and Metabolism. January 03, 2017

    It has been demonstrated that the neuropeptide oxytocin (OT) attenuates oxidative stress and inflammation in macrophages. In the current study, we examined the role of inflammation on the expression of the oxytocin receptor (OXTR). We hypothesized that OXTR expression is increased during the inflammation though a nuclear factor kappa B (NF-B) mediated pathway, thus responding as an acute phase protein. Inflammation was induced by treating macrophages (human primary, THP-1, and murine) with lipopolysaccharide (LPS) and monitored by expression of IL-6. Expression of OXTR and vasopressin receptors was assessed by qPCR and OXTR expression was confirmed by immunoblotting. Inflammation up-regulated OXTR transcription 10-250-fold relative to control in THP-1 and human primary macrophages, and increased OXTR protein expression. In contrast, vasopressin receptor-2 mRNA expression was reduced following LPS treatment. Blocking NF-B activation prevented the increase in OXTR transcription. OT treatment of control cells and LPS-treated cells increased ERK1/2 phosphorylation demonstrating activation of the OXTR/Gαq/11 signaling pathway. OT activation of OXTR reduced secretion of IL-6 in LPS-activated macrophages. Collectively, these findings suggest that OXTR is an acute phase protein, and that its increased expression is regulated by NF-B and functions to attenuate cellular inflammatory responses in macrophages.

    January 03, 2017   doi: 10.1152/ajpendo.00346.2016   open full text
  • Cardiometabolic risk improvement in response to a 3-yr lifestyle modification program in men: contribution of improved cardiorespiratory fitness vs weight loss.
    Borel, A.-L., Nazare, J.-A., Baillot, A., Almeras, N., Tremblay, A., Bergeron, J., Poirier, D. P., Despres, J.-P.
    AJP Endocrinology and Metabolism. December 27, 2016

    Objective: To examine the respective contributions of changes in visceral adiposity, subcutaneous adiposity, liver fat, and cardiorespiratory fitness (CRF) to the improvements in cardiometabolic risk markers observed in response to a 3-year healthy eating/physical activity lifestyle intervention. Research Design and Methods: Ninety-four out of 144 viscerally obese but otherwise healthy men completed a 3-year lifestyle intervention. Body weight, body composition and fat distribution were assessed by anthropometry and DEXA/computed tomography. CRF, adipokines, lipoprotein-lipid profile and 75g oral glucose tolerance were assessed. Results: CRF, visceral and subcutaneous adiposity significantly improved over the 3-year intervention, with a nadir at year 1 and a partial regain at year 3. Liver fat (estimated by insulin hepatic extraction) stabilized from year 1 to year 3 whereas HOMA-IR, ISI-Matsuda index and adiponectin continued to improve. Multivariate analysis revealed that visceral adiposity and estimated liver fat reductions both contributed to the improved ISI-Matsuda index observed over 3 years (R2=0.28, p<0.001). Three-year changes in fat mass and in CRF were independently associated with changes in visceral fat (adjusted R2=0.40, p<0.001) whereas only changes in CRF were associated with changes in estimated liver fat (adjusted R2=0.18, p<0.001). Conclusions: A long-term healthy eating/physical activity intervention in men improves several cardiometabolic risk markers over the long-term (3 years) despite a partial body weight regain observed between year 1 and year 3. The improvement in CRF contributes to visceral and estimated liver fat losses over the long term which in turn explain the benefits of the lifestyle intervention on cardiometabolic risk profile.

    December 27, 2016   doi: 10.1152/ajpendo.00278.2016   open full text
  • Reduced islet function contributes to impaired glucose homeostasis in fructose-fed mice.
    Asghar, Z. A., Cusumano, A., Yan, Z., Remedi, M. S., Moley, K. H.
    AJP Endocrinology and Metabolism. December 27, 2016

    Increased sugar consumption, particularly fructose, in the form of sweetened beverages and sweeteners in our diet adversely affects metabolic health. Because these effects are associated with features of the metabolic syndrome in humans, the direct effect of fructose on pancreatic islet function is unknown. We therefore examined the islet phenotype of mice fed excess fructose. Fructose-fed mice exhibited fasting hyperglycemia and glucose intolerance, but not hyperinsulinemia, dyslipidemia, or hyperuricemia. Islet function was impaired, with decreased glucose-stimulated insulin secretion and increased glucagon secretion, High fructose consumption led to alpha cell proliferation and upregulation of the fructose transporter GLUT5, which was localized only in alpha cells. Our studies demonstrate that excess fructose consumption contributes to hyperglycemia by affecting both beta and alpha cells of islets in mice.

    December 27, 2016   doi: 10.1152/ajpendo.00279.2016   open full text
  • Acute And Chronic Hyperglycemic Effects Of Vasopressin In Normal Rats, Involvement Of V1 A Receptors.
    Taveau, C., Chollet, C., Bichet, D. G., Velho, G., Guillon, G., Corbani, M., Roussel, R., Bankir, L., Melander, O., Bouby, N.
    AJP Endocrinology and Metabolism. December 20, 2016

    Recent epidemiological studies have revealed novel relationships between low water intake or high vasopressin (AVP) and the risk of hyperglycemia and diabetes. AVP V1A and V1B receptors (R) are expressed in liver and pancreatic islets, respectively. The present study was designed to determine the impact of different levels of circulating AVP on glucose homeostasis in normal Sprague Dawley rats, and the respective roles of V1AR and V1BR. We showed that acute injection of AVP induces a dose-dependent increase in glycemia. Pretreatment with a selective V1AR antagonist, but not a V1BR antagonist prevented dose-dependently the rise in glycemia. V1BR antagonism did not modify the hyperinsulinemic response resulting from AVP-induced hyperglycemia, but enhanced the fall in glucagonemia. Acute administration of selective V1AR or V1BR agonists confirmed the involvement of V1AR in the hyperglycemic effect of AVP. In chronic experiments, AVP levels were altered in both directions. Sustained AVP infusion through implantable minipumps induced a time-dependent increase in fasting glycemia, whereas lowering endogenous AVP by increasing water intake had no effect. After 4 weeks of AVP infusion, the rise in glycemia amounted to 1.1 mmol/L (p<0.01) without significant change in insulinemia. This effet was attenuated by cotreatment with a V1AR antagonist. Similar results were observed in lean Zucker rats. These findings demonstrate for the first time a causal link between chronic high AVP and hyperglycemia through V1AR activation and thus provide a pathophysiological explanation for the relationship observed in human cohorts between the AVP-hydration axis and the risk of diabetes.

    December 20, 2016   doi: 10.1152/ajpendo.00269.2016   open full text
  • Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity.
    Kocsis, T., Trencsenyi, G., Szabo, K., Baan, J. A., Muller, G., Mendler, L., Garai, I., Reinauer, H., Deak, F., Dux, L., Keller-Pinter, A.
    AJP Endocrinology and Metabolism. December 13, 2016

    The TGF-β family member myostatin (growth/differentiation factor-8, GDF-8) is a negative regulator of skeletal muscle growth. The hypermuscular Compact mice carry the 12-bp Mstn(Cmpt-dl1Abc) deletion in the sequence encoding the propeptide region of the precursor promyostatin and additional modifier genes of the Compact genetic background contribute to determine the full expression of the phenotype. In this study, by using mice strains carrying mutant or wild-type myostatin alleles with Compact genetic background, and non-mutant myostatin with wild-type background we studied separately the effect of the Mstn(Cmpt-dl1Abc) mutation or the Compact genetic background on morphology, metabolism and signaling. We show that both the Compact myostatin mutation and Compact genetic background account for determination of skeletal muscle size. Despite the increased musculature of Compacts, the absolute size of heart and kidney are not influenced by myostatin mutation; however, the Compact genetic background increases them. Both Compact myostatin and genetic background exhibit systemic metabolic effects. The Compact mutation decreases adiposity, improves whole body glucose uptake, insulin sensitivity and 18FDG uptake of skeletal muscle and white adipose tissue, while the Compact genetic background has opposite effect. Importantly, the mutation does not prevent the formation of mature myostatin; however, a decrease in myostatin level was observed leading to altered activation of Smad2, Smad1/5/8 and Akt, and increased level of pAS160, a Rab-GTPase activating protein responsible for GLUT4 translocation. Based on our analysis the Compact genetic background strengthens the effect of myostatin mutation on muscle mass, but can compensate each other when systemic metabolic effects are compared.

    December 13, 2016   doi: 10.1152/ajpendo.00216.2016   open full text
  • Effects of paternal obesity on growth and adiposity of male rat offspring.
    Lecomte, V., Maloney, C. A., Wang, K. W., Morris, M. J.
    AJP Endocrinology and Metabolism. December 13, 2016

    Emerging evidence suggests that paternal obesity plays an important role in offspring health. We previously showed that female offspring from high fat diet (HFD) fed male rats develop glucose intolerance due to impaired insulin secretion. Here, we focussed on the health outcomes of male offspring from HFD fed fathers. Male Sprague Dawley rats (3 week-old) were fed control diet (CD-F0) or HFD (HFD-F0) for 12 weeks before mating with control fed females. Male offspring fed control diet were studied at 8 weeks and 6 months. While male offspring from HFD-F0 did not develop any obvious glucose metabolism defects, a growth deficit phenotype was observed from birth onwards. Male offspring from HFD-F0 had reduced birth weight compared to CD-F0, followed by reduced post-weaning growth from 9 weeks of age. This resulted in 10% reduction in body weight at 6 months with significantly smaller fat pads and muscles. Reduced circulating levels of Growth Hormone (GH) and IGF1 were detected at 8 weeks and 6 months. Expression of adipogenesis markers was decreased in adipose tissue of HFD-F0 offspring at 8 weeks and 6 months, as well as expression of growth markers in muscle of HFD-F0 offspring at 8 weeks. We propose that the reduced GH secretion at 8 weeks of age altered the growth of male offspring from HFD-F0, resulting in smaller animals from 9 weeks to 6 months of age. Further, disturbance in muscle lipid metabolism genes was observed in HFD-F0 offspring, potentially increasing their metabolic risk.

    December 13, 2016   doi: 10.1152/ajpendo.00262.2016   open full text
  • Postnatal Treatment with Metyrapone Attenuates the Effects of Diet-Induced Obesity in Female Rats Exposed to Early Life Stress.
    Murphy, M. O., Herald, J. B., Wills, C. T., Unfried, S. G., Cohn, D. M., Loria, A. S.
    AJP Endocrinology and Metabolism. December 13, 2016

    This study tested the hypothesis that female rats pups exposed to maternal separation (MatSep), a model of early life stress, display an exacerbated response to diet-induced obesity compared to male rats. Also, we tested whether postnatal treatment with metyrapone (MTP), a corticosterone synthase inhibitor, would attenuate this phenotype. MatSep was performed in WKY offspring by separation from the dam (3 hr/day, postnatal day 2-14). Upon weaning, male and female rats were placed on regular chow (ND, 18% kcal fat) or HFD (60% kcal fat). Non-disturbed littermates served as controls. In male rats, no diet-induced differences in body weight (BW), glucose tolerance, or fat pad weight and morphology were observed between MatSep and control male rats. However, female MatSep rats displayed increased BW gain, fat pad weights and glucose intolerance compared to control rats (p<0.05). Also, HFD increased plasma corticosterone and leptin levels (p<0.05) in female MatSep compared to control rats while insulin and adiponectin levels were similar between groups. Female control and MatSep offspring were treated with MTP (50 µg/g i.p.), 30 minutes prior to the daily separation. MTP treatment significantly attenuated diet-induced obesity risk factors including elevated adiposity, hyperleptinemia and glucose intolerance. These findings show that exposure to stress hormones during early life could be a key event to enhance diet-induced obesity and metabolic disease in female rats. Thus, pharmacological and/or behavioral inflection of the stress levels is a potential therapeutic approach for prevention of early life stress-enhanced obesity and metabolic disease.

    December 13, 2016   doi: 10.1152/ajpendo.00308.2016   open full text
  • Prior Lactation Reduces Future Diabetic Risk through Sustained Post-Weaning Effects on Insulin Sensitivity.
    Bajaj, H., Ye, C., Hanley, A. J., Connelly, P. W., Sermer, M., Zinman, B., Retnakaran, R.
    AJP Endocrinology and Metabolism. December 13, 2016

    Breastfeeding ≥12 months is recommended for optimal infant nutrition but may hold maternal benefits, as well. Indeed, lactation has been associated with lower long-term risk of diabetes in the mother but the mechanism by which it imparts sustained post-weaning effects on glucose tolerance remains unclear. In this context, we postulated that lactation potentially could induce post-weaning beneficial effects on glucose tolerance by modifying the natural history of insulin sensitivity and/or pancreatic beta-cell function over time. Thus, in this study, we evaluated the relationships between duration of lactation (≤3 months (n=70), 3-12 months (n=140), ≥12 months (n=120)) and trajectories of insulin sensitivity/resistance, beta-cell function, and glycemia over the first 3-years postpartum in a cohort of 330 women comprising the full spectrum of glucose tolerance in pregnancy, who underwent serial metabolic characterization, including oral glucose tolerance tests, at 3-months, 1-year, and 3-years postpartum. The prevalence of dysglycemia (pre-diabetes/diabetes) at 3-years postpartum was lower in women who breastfed ≥12 months (12.5%) than in those who breastfed ≤3 months (21.4%) or for 3-12 months (25.7%)(overall P=0.028). On logistic regression analysis, lactation ≥12 months independently predicted a lower likelihood of pre-diabetes/diabetes at 3-years postpartum (OR=0.37, 95%CI 0.18-0.78, P=0.009). Notably, lactation ≥12 months predicted lesser worsening of insulin sensitivity/resistance (P<0.0001), fasting glucose (P<0.0001), and 2-hour glucose (P=0.011) over 3-years, as compared to lactation ≤3 months, but no differences in beta-cell function (P≥0.37). It thus emerges that adherence to current breastfeeding recommendations reduces future diabetic risk through sustained post-weaning effects on insulin sensitivity/resistance but not beta-cell function.

    December 13, 2016   doi: 10.1152/ajpendo.00403.2016   open full text
  • UCP1 Inhibition in Cidea-Overexpressing Mice is Physiologically Counteracted by Brown Adipose Tissue Hyperrecruitment.
    Fischer, A. W., Shabalina, I. G., Mattsson, C. L., Abreu-Vieira, G., Cannon, B., Nedergard, J., Petrovic, N.
    AJP Endocrinology and Metabolism. December 06, 2016

    Cidea is a gene highly expressed in thermogenesis-competent (UCP1-containing) adipose cells, both brown and brite/beige. Here we initially demonstrate a remarkable adipose-depot specific regulation of Cidea expression. In classical brown fat, Cidea mRNA is expressed continuously and invariably, irrespective of tissue recruitment. However, Cidea protein levels are regulated posttranscriptionally, being conspicuously induced in the thermogenically recruited state. In contrast, in brite fat, Cidea protein levels are regulated at the transcriptional level, and Cidea mRNA and protein levels are proportional to tissue "briteness". Although routinely followed as a thermogenic molecular marker, Cidea function is not clarified. Here we employed a gain-of-function approach to examine a possible role of Cidea in the regulation of thermogenesis. We utilized transgenic aP2-hCidea mice that overexpress human Cidea in all adipose tissues. We demonstrate that UCP1 activity is markedly suppressed in brown-fat mitochondria isolated from aP2-hCidea mice. However, mitochondrial UCP1 protein levels were identical in wild-type and transgenic mice. This implies a regulatory effect of Cidea on UCP1, but as we demonstrate that Cidea itself is not localized to mitochondria, we propose an indirect inhibitory effect. The Cidea-induced inhibition of UCP1 activity is physiologically relevant, since the mice, through an appropriate homeostatic compensatory mechanism, increased the total amount of UCP1 in the tissue to exactly match the diminished thermogenic capacity of the UCP1 protein and retain unaltered nonshivering thermogenic capacity. Thus, we verified Cidea as being a marker of thermogenesis-competent adipose tissues, but we conclude that Cidea, unexpectedly, functions molecularly as an indirect inhibitor of thermogenesis.

    December 06, 2016   doi: 10.1152/ajpendo.00284.2016   open full text
  • Role Of Autonomic Nervous System In Chronic Cns Mediated Antidiabetic Action Of Leptin.
    da Silva, A. A., Hall, J. E., Moak, S. P., Browning, J., Houghton, H. J., Micheloni, G. C., do Carmo, J. M.
    AJP Endocrinology and Metabolism. December 06, 2016

    This study tested whether chronic ganglionic blockade or hepatic vagotomy attenuates the chronic CNS-mediated antidiabetic and cardiovascular effects of leptin. Male Sprague-Dawley rats were instrumented with telemetry probes and arterial and venous catheters for determination of blood pressure (BP), heart rate (HR), blood sampling and intravenous (IV) infusions. An intracerebroventricular (ICV) cannula was placed into the brain lateral ventricle for infusion of leptin or vehicle. After control measurements, streptozotocin (STZ) was injected IV (50 mg/kg) to induce diabetes and 5 days later leptin (n=6) or saline vehicle (n=5) was infused ICV for 12 days via osmotic pumps. Beginning on day 6 of leptin treatment, the ganglionic blocker hexamethonium (15 mg/kg/day, IV) was infused, while leptin infusion was continued, to assess the role of the autonomic nervous system. Induction of diabetes was associated with increases in blood glucose (98±7 to 350±19 mg/dL), food intake (23±3 to 43±3 g/d), decreases in heart rate (-70±11 bpm), polyuria, and increased water consumption, which were all completely normalized by ICV leptin infusion. Although hexamethonium attenuated leptin's effect on HR, it failed to impair leptin's ability to restore euglycemia or to prevent the polyuria or increased water intake in STZ-diabetic rats. We also found that after pretreatment with hexamethonium (n=8), ICV leptin infusion, during continued ganglionic blockade, completely normalized blood glucose in diabetic rats. In addition, selective hepatic vagotomy did not attenuate leptin's ability to restore euglycemia in diabetic rats. These results suggest that leptin's powerful chronic CNS antidiabetic actions are mediated primarily via non-autonomic mechanisms.

    December 06, 2016   doi: 10.1152/ajpendo.00301.2016   open full text
  • Long-term high-fat diet induces hippocampal microvascular insulin resistance and cognitive dysfunction.
    Fu, Z., Wu, J., Nesil, T., Li, M. D., Aylor, K. W., Liu, Z.
    AJP Endocrinology and Metabolism. November 29, 2016

    Insulin action on hippocampus improves cognitive function and obesity and type 2 diabetes are associated with decreased cognitive function. Cerebral microvasculature plays a critical role in maintaining cerebral vitality and function by supplying nutrients, oxygen and hormones such as insulin to cerebral parenchyma including hippocampus. In skeletal muscle insulin actively regulates microvascular opening and closure and this action is impaired in the insulin resistant states. To examine insulin's action on hippocampal microvasculature and parenchyma and the impact of diet-induced obesity we determined cognitive function, and microvascular insulin responses, parenchyma insulin responses and capillary density in the hippocampus in 2-month and 8-month rats on chow diet and 8-month rats on long-term high-fat diet (6 months). Insulin infusion increased hippocampal microvascular perfusion in rats on chow diet by ~80-90%. High-fat diet feeding completely abolished insulin-mediated microvascular responses and protein kinase B phosphorylation but did not alter the capillary density in the hippocampus. This was associated with a significantly decreased cognitive function assessed using both the two-trial spontaneous alternation behavior test and the novel object recognition test. As the microvasculature provides the needed endothelial surface area for delivery of nutrients, oxygen and insulin to hippocampal parenchyma, we conclude that hippocampal microvascular insulin resistance may play a critical role in the development of cognitive impairment seen in obesity and diabetes. Our results suggest that improvement in hippocampal microvascular insulin sensitivity might help improve or reverse cognitive function in the insulin resistant states.

    November 29, 2016   doi: 10.1152/ajpendo.00297.2016   open full text
  • High fat diet-induced obesity regulates MMP3 to modulate depot- and sex-dependent adipose expansion in C57BL/6J mice.
    Wu, Y., Lee, M.-J., Ido, Y., Fried, S. K.
    AJP Endocrinology and Metabolism. November 22, 2016

    Increased adipocyte size is hypothesized to signal the recruitment of adipose progenitor cells (APCs) to expand tissue storage capacity. To investigate depot- and sex-differences in adipose growth, male and female C57BL/6J mice (10-weeks-old) were challenged with high fat (HF) or low fat (LF) diets (D) for 14 weeks. The HFD increased gonadal (GON) depot weight by adipocyte hypertrophy and hyperplasia in females, but hypertrophy alone in males. In both sexes, inguinal (ING) adipocytes were smaller than GON and depot expansion was due to hypertrophy. Matrix metalloproteinase 3 (Mmp3), an anti-adipogenic factor, and its inhibitors, Timps modulate the extracellular matrix remodeling needed for depot expansion. Mmp3 mRNA was depot different (ING>GON), higher in females than males and mainly expressed in APCs. In males, HFD-induced obesity increased tissue and APC Mmp3 mRNA levels and MMP3 protein and enzymatic activity. In females however, HFD significantly decreased MMP3 protein without affecting its mRNA levels. MMP3 activity also decreased (significant in ING). Timp4 mRNA was mainly expressed in adipocytes, and HFD-induced obesity tended to increase the ratio of TIMP4 to MMP3 protein in females, while decreasing it in males. Overexpression of Mmp3 in 3T3-L1 preadipocytes or rhMMP3 protein added to primary human preadipocytes inhibited differentiation, while rhTIMP4 improved adipogenesis and attenuated the inhibitory effect of rhMMP3. These data suggest that HFD-induced obesity downregulates APC MMP3 expression to trigger adipogenesis and adipocyte TIMP4 may modulate this process to regulate hyperplastic vs. hypertrophic adipose tissue expansion, fat distribution and metabolic health in a sex- and depot-dependent manner.

    November 22, 2016   doi: 10.1152/ajpendo.00128.2016   open full text
  • Glucosamine induces ER stress by disrupting lipid-linked oligosaccharide biosynthesis and N-linked protein glycosylation.
    Beriault, D. R., Dang, V. T., Zhong, L. H., Petlura, C. I., McAlpine, C. S., Shi, Y., Werstuck, G. H.
    AJP Endocrinology and Metabolism. November 22, 2016

    Glucosamine is an essential substrate for N-linked protein glycosylation. However, elevated levels of glucosamine can induce endoplasmic reticulum (ER) stress. Glucosamine-induced ER stress has been implicated in the development of diabetic complications including atherosclerosis and hepatic steatosis. In this study, we investigate the potential relationship between the effects of glucosamine on lipid linked oligosaccharide (LLO) biosynthesis, N-linked glycosylation, and ER homeostasis. Mouse embryonic fibroblasts (MEFs) were cultured in the presence of 0-5 mM glucosamine for up to 18 hours and LLO biosynthesis was monitored by fluorescence-assisted carbohydrate electrophoresis. ER stress was determined by quantification of unfolded protein response (UPR) gene expression. We found that exposure of MEFs to ≥1 mM glucosamine significantly impaired the biosynthesis of mature (Glc3Man9GlcNAc2) LLOs prior to the activation of the UPR, which resulted in the accumulation of an LLO intermediate (Man3GlcNAc2). Addition of 4-phenylbutyric acid (4PBA), a chemical chaperone, was able to alleviate ER stress but did not rescue LLO biosynthesis. Other ER stress inducing agents, including dithiothreitol and thapsigargin, had no effect on LLO levels. Together, these data suggest that elevated concentrations of glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. We hypothesize that this pathway represents a causative link between hyperglycemia and the development of diabetic complications.

    November 22, 2016   doi: 10.1152/ajpendo.00275.2016   open full text
  • Loss of Glutaredoxin 3 Impedes Mammary Lobuloalveolar Development during Pregnancy and Lactation.
    Pham, K., Dong, J., Jiang, X., Qu, Y., Yu, H., Yang, Y., Olea, W., Marini, J. C., Chan, L., Wang, J., Wehrens, X. H. T., Cui, X., Li, Y., Hadsell, D. L., Cheng, N.
    AJP Endocrinology and Metabolism. November 15, 2016

    Mammalian glutaredoxin 3 (Grx3) has been shown to be important for regulating cellular redox homeostasis in the cell. Our previous studies indicate that Grx3 is significantly overexpressed in various human cancers including breast cancer and demonstrate that Grx3 controls cancer cell growth and invasion by regulating ROS and NF-B signaling pathways. However, it remains to be determined whether Grx3 is required for normal mammary gland development and how it contributes to epithelial cell proliferation and differentiation in vivo. In the present study, we examined Grx3 expression in different cell types within the developing mouse mammary gland (MG) and found enhanced expression of Grx3 at pregnancy and lactation stages. To assess the physiological role of Grx3 in MG, we generated the mutant mice in which Grx3 was deleted specifically in mammary epithelial cells (MECs). Although the reduction of Grx3 expression had only minimal effects on mammary ductal development in virgin mice, it did reduce alveolar density during pregnancy and lactation. The impairment of lobuloalveolar development was associated with high levels of ROS accumulation and reduced expression of milk protein genes. In addition, proliferative gene expression was significantly suppressed with proliferation defects occurring in knockout MECs during alveolar development compared to wild type controls. Therefore, our findings suggest that Grx3 is a key regulator of ROS in vivo and is involved in pregnancy-dependent mammary gland development and secretory activation through modulating cellular ROS.

    November 15, 2016   doi: 10.1152/ajpendo.00150.2016   open full text
  • Dietary gossypol suppressed postprandial TOR signaling and elevated ER stress pathways in Turbot (Scophthalmus maximus L.).
    Bian, F., Jiang, H., Man, M., Mai, K., Zhou, H., Xu, W., He, G.
    AJP Endocrinology and Metabolism. November 15, 2016

    Gossypol is known to be a polyphenolic compound toxic to animals. However, its molecular targets are far from fully characterized. To evaluate the physiological and molecular effects of gossypol, we chose turbot (Scophthalmus maximus L.), a carnivorous fish as our model species. Juvenile turbots (7.83 ± 0.02 g) were fed with diets containing gradient levels of gossypol at 0 (G0), 600 (G1) and 1,200 (G2) mg/kg diets for 11 weeks. After the feeding trial, fish growth, body protein and fat contents were significantly reduced in G2 group compared with those of G0 group (P< 0.05). Gossypol impacted little on digestive enzyme activities and intestine morphology. However, gossypol caused liver fibrosis and stimulated chemokine and pro-inflammatory cytokine secretions. More importantly, gossypol suppressed target of rapamycin (TOR) signaling and induced endoplasmic reticulum (ER) stress pathway in both feeding experiment and cell cultures. Our results demonstrated that gossypol inhibited TOR signaling and elevated ER stress pathways both in vivo and in vitro, thus providing new mechanism of action of gossypol in nutritional physiology.

    November 15, 2016   doi: 10.1152/ajpendo.00285.2016   open full text
  • Evaluation of CSF and Plasma Biomarkers of Brain Melanocortin Activity in Response to Caloric Restriction in Humans.
    Page-Wilson, G., Nguyen, K. T., Atalayer, D., Meece, K., Bainbridge, H. A., Korner, J., Gordon, R. J., Panigrahi, S. K., White, A., Smiley, R., Wardlaw, S. L.
    AJP Endocrinology and Metabolism. November 15, 2016

    The melanocortin neuronal system, comprised of hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, is a leptin target that regulates energy balance and metabolism, but studies in humans are limited by lack of reliable biomarkers for brain melanocortin activity. The objective of this study was to measure the POMC prohormone and its processed peptide, ß-endorphin (ß-EP), in cerebrospinal fluid (CSF) and AgRP in CSF and plasma after calorie restriction to validate their utility as biomarkers of brain melanocortin activity. CSF and plasma were obtained from 10 lean and obese subjects after fasting (40h) and refeeding (24h) and from 8 obese subjects before and after 6-weeks of dieting (800 kcal/day) to assess changes in neuropeptide and hormone levels. After fasting, plasma leptin decreased to 35% and AgRP increased to 153% of baseline. During refeeding AgRP declined as leptin increased; CSF ß-EP increased but POMC did not change. Relative changes in plasma and CSF leptin were blunted in obese subjects. After dieting, plasma and CSF leptin decreased to 46% and 70% of baseline; CSF POMC and ß-EP decreased; plasma AgRP increased. At baseline AgRP correlated negatively with insulin and HOMA-IR and positively with the Matsuda index. Thus following chronic calorie restriction POMC and ß-EP declined in CSF while acutely only ß-EP changed. Plasma AgRP, however, increased after both acute and chronic restriction. These results support the use of CSF POMC and plasma AgRP as biomarkers of hypothalamic melanocortin activity and provide evidence linking AgRP to insulin sensitivity.-

    November 15, 2016   doi: 10.1152/ajpendo.00330.2016   open full text
  • Obesity-associated extracellular mtDNA activates central TGF{beta} pathway to cause blood pressure increase.
    Ale, A., Zhang, Y., Han, C., Cai, D.
    AJP Endocrinology and Metabolism. November 15, 2016

    Hypothalamic inflammation was recently found to mediate obesity-related hypertension, but the responsible upstream mediators remain unexplored. In this study, we show that dietary obesity is associated with extracellular release of mitochondrial DNA (mtDNA) into the cerebrospinal fluid, and that central delivery of mtDNA mimics TGFβ excess to activate downstream signaling pathways. Physiological study reveals that central administration of mtDNA or TGFβ are both sufficient to cause hypertension in mice. Knockout of the TGFβ receptor in pro-opiomelanocortin neurons counteracts the hypertensive effect of not only TGFβ but mtDNA excess, while the hypertensive action of central mtDNA can be blocked pharmacologically by a TGFβ receptor antagonist or genetically by TGFβ receptor knockout. Finally, we confirm that obesity-induced hypertension can be reversed through central treatment with TGFβ receptor antagonist. In conclusion, circulating mtDNA in the brain employs neural TGFβ pathway to mediate a central inflammatory mechanism of obesity-related hypertension.

    November 15, 2016   doi: 10.1152/ajpendo.00337.2016   open full text
  • Light-load resistance exercise increases muscle protein synthesis and hypertrophy signaling in elderly men.
    Agergaard, J., Bulow, J., Jensen, J. K., Reitelseder, S., Drummond, M. J., Schjerling, P., Scheike, T., Serena, A., Holm, L.
    AJP Endocrinology and Metabolism. November 15, 2016

    Introduction. The present study investigated whether well-tolerated light-load resistance exercise (LL-RE) affects skeletal muscle fractional synthetic rate (FSR) and anabolic intracellular signaling as a way to counteract age-related loss of muscle mass. Methods. Untrained healthy men (age: +65 yrs) were subjected to 13 hours supine rest. After 21/2 hours of rest, unilateral LL-RE was conducted consisting of leg extensions (10 sets, 36 repetitions) at 16% 1RM. Subsequently, the subjects were randomized to oral intake of PULSE (4g whey protein/hour; N=10), BOLUS (28g whey protein at 0 hours and 12g whey protein at 7 hours post-exercise; N=10) or placebo (4g maltodextrin/hour; N=10). Quadriceps muscle biopsies were taken at 0, 3, 7 and 10 hours post-exercise from both the resting and exercised leg. Myofibrillar-FSR and activity of select targets from the mTORC1-signalling cascade were analyzed from the biopsies. Results. LL-RE increased myofibrillar-FSR compared to the resting leg throughout the 10h post-exercise period. The p-AKT (T308) expression increased in the exercise leg immediately after exercise. This increase persisted in the placebo group only. Levels of p-4E-BP1 (T37/46) increased throughout the post-exercise period in the exercised leg in the placebo and BOLUS group and peaked at 7h. In all three groups, p-eEF2 (T56) decreased in response to LL-RE. Conclusion. We conclude that resistance exercise at only 16% 1RM increased myofibrillar-FSR, irrespective of nutrient type and feeding pattern, which indicates an anabolic effect of LL-RE in elderly individuals. This finding was supported by increased signaling for translation initiation and translation elongation in response to LL-RE.

    November 15, 2016   doi: 10.1152/ajpendo.00164.2016   open full text
  • Citrulline directly modulates muscle protein synthesis via the PI3K/ MAPK/4E-BP1 pathways: Evidence from in vivo, ex vivo and in vitro studies.
    Le Plenier, S., Goron, A., Sotiropoulos, A., Archambault, E., Guihenneuc, C., Walrand, S., Salles, J., Jourdan, M., Neveux, N., Cynober, L., Moinard, C.
    AJP Endocrinology and Metabolism. November 08, 2016

    Citrulline (CIT) is an endogenous amino acid produced by the intestine. Recent literature consistently shows CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both inter-organ and intra-organ trafficking and metabolism, we combined three approaches: in vivo, ex vivo and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (p < 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, p < 0.05) and the CIT effect was abolished in the presence of wortmannin, rapamycin and PD98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD98059 inhibited the CIT effect on S6K1, whereas only LY294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control.

    November 08, 2016   doi: 10.1152/ajpendo.00203.2016   open full text
  • Kupffer Cells Facilitate the Acute Effects of Leptin on Hepatic Lipid Metabolism.
    Metlakunta, A., Huang, W., Stefanovic-Racic, M., Dedousis, N., Sipula, I., O'Doherty, R. M.
    AJP Endocrinology and Metabolism. November 08, 2016

    Leptin has potent effects on lipid metabolism in a number of peripheral tissues. In liver, an acute leptin infusion (~120-min) stimulates hepatic fatty acid oxidation (~30%) and reduces triglycerides (TG, ~40%), effects that are dependent on PI3-kinase (PI3K) activity. In the current study we addressed the hypothesis that leptin actions on liver-resident immune cells are required for these metabolic effects. Myeloid cell-specific deletion (My) of the leptin receptor (ObR) in mice or depletion of liver Kupffer cells (KC) in rats in vivo prevented the acute effects of leptin on liver lipid metabolism, while the metabolic effects of leptin were maintained in mice lacking ObR in hepatocytes (HepObR). Notably, liver TG were elevated in both lean and obese MYObR, but the degree of obesity and insulin resistance induced by a high fat diet was similar to control mice. In isolated primary hepatocytes (HEP), leptin had no effects on HEP lipid metabolism and only weakly stimulated PI3K. However, the co-culture of KC with HEP restored leptin action on HEP fatty acid metabolism and stimulation of HEP PI3K. Notably, leptin stimulated the release from KC of a number of cytokines. However, the exposure of HEP to these cytokines individually (GM-CSF, IL-1α, IL-1β, IL-6, IL-10, IL-18) or in combination had no effects on HEP lipid metabolism. Together, these data demonstrate a role for liver mononuclear cells in the regulation of liver lipid metabolism by leptin

    November 08, 2016   doi: 10.1152/ajpendo.00250.2016   open full text
  • Chronic hyperprolactinemia evoked by disruption of lactotrope dopamine D2 receptors impacts on liver and adipocyte genes related to glucose and insulin balance.
    Luque, G. M., Lopez-Vicchi, F., Ornstein, A. M., Brie, B., De Winne, C., Fiore, E., Perez Millan, M. I., Mazzolini, G., Rubinstein, M., Becu-Villalobos, D.
    AJP Endocrinology and Metabolism. November 01, 2016

    We studied the impact of high prolactin titers on liver and adipocyte gene expression related to glucose and insulin homeostasis, in correlation with obesity onset. To that end we used mutant female mice that selectively lack dopamine type 2 receptors (D2Rs) from pituitary lactotropes (lacDrd2KO) which have chronic high prolactin levels associated with increased body weight, marked increments in fat depots, adipocyte size, and serum lipids, a metabolic phenotype which intensifies with age. LacDrd2KO mice of two developmental ages, 5 and 10 months were used. In the first time point, obesity and increased body weight are marginal even though mice are hyperprolactinemic, while at 10 months there is marked adiposity with a 136 % increase in gonadal fat, and a 36 % increase in liver weight due to lipid accumulation. LacDrd2KO mice had glucose intolerance, hyperinsulinemia, and impaired insulin response to glucose, already in early stages of obesity, but changes in liver and adipose tissue transcription factors were time and tissue dependent. In chronic hyperprolactinemic mice liver Prlr were upregulated, there was liver steatosis, altered expression of the lipogenic transcription factor Chrebp, and blunted response of Srebp-1c to refeeding at 5 months of age, while no effect was observed in the glycogenesis pathway. On the other hand, in adipose tissue a marked decrease in lipogenic transcription factor expression was observed when morbid obesity was already settled. These adaptive changes underscore the role of prolactin signaling in different tissues to promote energy storage

    November 01, 2016   doi: 10.1152/ajpendo.00200.2016   open full text
  • The role of dietary fat in obesity-induced insulin resistance.
    Lackey, D. E., Lazaro, R. G., Li, P., Johnson, A., Hernandez-Carretero, A., Weber, N., Vorobyova, I., Tsukomoto, H., Osborn, O.
    AJP Endocrinology and Metabolism. November 01, 2016

    Consumption of excess calories results in obesity and insulin resistance and has been intensively studied in mice and humans. The objective of this study was to determine the specific contribution of dietary fat rather than total caloric intake to the development of obesity associated insulin resistance. We used an intragastric feeding method to overfeed excess calories from low fat diet (and isocalorically matched high fat diet) through a surgically implanted gastric feeding tube to generate obesity in wild-type mice followed by hyperinsulinemic-euglycemic clamp studies to assess the development of insulin resistance. We show that overfeeding low fat diet results in similar levels of obesity as high fat diet feeding in mice. However, despite a similar body weight, obese high fat diet fed mice are more insulin resistant than mice fed an isocaloric low fat diet. Therefore, increased proportion of calories from dietary fat further potentiates insulin resistance in the obese state. Furthermore, crossover diet studies revealed that reduction in dietary fat composition improves glucose tolerance in obesity. In the context of the current obesity and diabetes epidemic it is particularly important to fully understand the role of dietary macronutrients in the potentiation and amelioration of disease.

    November 01, 2016   doi: 10.1152/ajpendo.00323.2016   open full text
  • Fourth ventricle leptin infusions dose dependently activate hypothalamic signal transducer and activator of transcription 3 (STAT3).
    Harris, R. B. S., Desai, B. N.
    AJP Endocrinology and Metabolism. November 01, 2016

    Previous studies have shown that very low dose infusions of leptin into the 3rd or the 4th ventricle alone have little effect on energy balance, but simultaneous low dose infusions cause rapid weight loss and increased phosphorylation of STAT3 (pSTAT3) in hypothalamic sites that express leptin receptors. Other studies show that injecting high doses leptin into the 4th ventricle inhibits food intake and weight gain. Therefore, we tested whether 4th ventricle leptin infusions that cause weight loss are associated with increased leptin signaling in the hypothalamus. In a dose response study 14 day infusions of increasing doses of leptin showed significant hypophagia, weight loss and increased hypothalamic pSTAT3 in rats receiving at least 0.9 μg leptin/day. In a second study 0.6 μg leptin/day transiently inhibited food intake and reduced carcass fat, but had no significant effect on energy expenditure. In a final study we identified the localization of STAT3 activation in the hypothalamus of rats receiving 0, 0.3 or 1.2 μg leptin/day. The high dose of leptin, which caused weight loss in the first experiment, increased pSTAT3 in the ventromedial, dorsomedial and arcuate nuclei of the hypothalamus. The low dose which increased brown fat UCP 1, but did not affect body composition in the first experiment, had little effect on hypothalamic pSTAT3. We propose that hindbrain leptin increases the precision of control of energy balance by lowering the threshold for leptin signaling in the forebrain. Further studies are needed to directly test this hypothesis.

    November 01, 2016   doi: 10.1152/ajpendo.00343.2016   open full text
  • Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of L-DOPA.
    Jiang, X., Zhang, Y., Yang, Y., Yang, J., Asico, L. D., Chen, W., Felder, R. A., Armanado, I., Jose, P. A., Yang, Z.
    AJP Endocrinology and Metabolism. October 25, 2016

    Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of L-DOPA, via the L-type amino acid transporter (LAT) at the plasma membrane. The uptake of L-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. L-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than salt-resistant BALB/c mice. The deficient renal cortical uptake of L-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension.

    October 25, 2016   doi: 10.1152/ajpendo.00116.2016   open full text
  • Activation of {beta}3-adrenoceptors increases in vivo free fatty acid uptake and utilization in brown but not white fat depots in high-fat fed rats.
    Warner, A., Kjellstedt, A., Carreras, A., Bottcher, G., Peng, X.-R., Seale, P., Oakes, N., Linden, D.
    AJP Endocrinology and Metabolism. October 25, 2016

    Activation of brown adipose tissue (BAT) and browning of white adipose tissue (WAT) present potential new therapies for obesity and type 2 diabetes. Here, we examined the effects of β3-adrenergic stimulation on tissue-specific uptake and storage of free fatty acids (FFA) and its implications for whole-body FFA metabolism in diet-induced obese rats using a multi-radiotracer technique. Male Wistar rats were high fat-fed for 12 weeks and administered β3-agonist CL316,243 (CL, 1 mg/kg/day) or saline via osmotic minipumps during the last three weeks. The rats were then fasted and acutely infused with a tracer mixture (14C-palmitate and the partially-metabolized 3H-R-bromopalmitate) under anesthesia. CL infusion decreased body weight gain and fasting plasma glucose levels. While core body temperature was unaffected, infrared thermography showed an increase in tail heat dissipation following CL infusion. Interestingly, CL markedly increased both FFA storage and utilization in interscapular and perirenal BAT while the flux of FFA to skeletal muscle was decreased. In this rat model of obesity, only sporadic populations of beige adipocytes were detected in the epididymal WAT depot of CL-infused rats and there was no change in FFA uptake or utilization in WAT following CL infusion. In summary, β3-agonism robustly increased FFA flux to BAT coupled with enhanced utilization. Increased BAT activation most likely drove the increased tail heat dissipation to maintain thermostasis. Our results emphasize the quantitative role of brown fat as the functional target of β3-agonism in obesity.

    October 25, 2016   doi: 10.1152/ajpendo.00204.2016   open full text
  • Adult Expression of PGC-1{alpha} and {beta} in Skeletal Muscle is Not Required for Endurance Exercise-Induced Enhancement of Exercise Capacity.
    Ballmann, C., Tang, Y., Bush, Z., Rowe, G. C.
    AJP Endocrinology and Metabolism. October 25, 2016

    Exercise has been shown to be the best intervention in the treatment of many diseases. Many of the benefits of exercise are mediated by adaptions induced in skeletal muscle. The peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family of transcriptional coactivators has emerged as being key mediators of the exercise response and is considered to be essential for many of the adaptions seen in skeletal muscle. However, the contribution of the PGC-1s in skeletal muscle has been evaluated by the use of either whole body or congenital skeletal muscle-specific deletion. In these models, PGC-1s were never present, thereby opening the possibility to developmental compensation. Therefore, we generated an inducible muscle-specific deletion of PGC-1α and β (iMyo-PGC-1DKO), in which both PGC-1α and β can be deleted specifically in adult skeletal muscle. These iMyo-PGC-1DKO animals were used to assess the role of both PGC-1α and β in adult skeletal muscle and their contribution to the exercise training response. Untrained iMyo-PGC-1DKO animals exhibited a time-dependent decrease in exercise performance 8 weeks post deletion, similar to what was observed in the congenital muscle-specific PGC-1DKOs. However, after 4 weeks of voluntary training, the iMyo-PGC-1DKOs exhibited an increase in exercise performance with a similar adaptive response compared to control animals. This increase was associated with an increase in electron transport complex (ETC) expression and activity in the absence of PGC-1α and β expression. Taken together these data suggest that PGC-1α and β expression are not required for training-induced exercise performance, highlighting the contribution of PGC-1 independent mechanisms.

    October 25, 2016   doi: 10.1152/ajpendo.00209.2016   open full text
  • Pre-sleep protein ingestion does not compromise the muscle protein synthetic response to protein ingested the following morning.
    Wall, B. T., Burd, N. A., Franssen, R., Gorissen, S. H., Snijders, T., Senden, J. M., Gijsen, A. P., van Loon, L. J. C.
    AJP Endocrinology and Metabolism. October 25, 2016

    Protein ingestion before sleep augments post-exercise muscle protein synthesis during overnight recovery. Purpose: It is unknown whether post-exercise and pre-sleep protein consumption modulates post-prandial protein handling and myofibrillar protein synthetic responses the following morning. Sixteen healthy young (24±1 y) men performed unilateral resistance-type exercise (contralateral leg acting as a resting control) at 20:00 h. Participants ingested 20 g protein immediately after exercise plus 60 g protein pre-sleep (PRO group; n=8) or equivalent boluses of carbohydrate (CON; n=8). The subsequent morning participants received primed-continuous infusions of L-[ring-2H5]phenylalanine and L-[1-13C]leucine combined with ingestion of 20 g intrinsically L-[1-13C]phenylalanine and L-[1-13C]leucine labelled protein to assess postprandial protein handling and myofibrillar protein synthesis in the rested and exercised leg in CON and PRO. Exercise increased post-absorptive myofibrillar protein synthesis rates the subsequent day (P<0.001), with no differences between treatments. Protein ingested in the morning increased myofibrillar protein synthesis in both the exercised- and rested-leg (P<0.01), with no differences between treatments. Myofibrillar protein bound L-[1-13C]phenylalanine enrichments were greater in the exercised (0.016±0.002 and 0.015±0.002 MPE in CON and PRO, respectively) versus rested (0.010±0.002 and 0.009±0.002 MPE in CON and PRO, respectively) leg (P<0.05), with no differences between treatments (P>0.05). The additive effects of resistance-type exercise and protein ingestion on myofibrillar protein synthesis persist for >12 h after exercise and are not modulated by protein consumption during acute post-exercise recovery. This work provides evidence of an extended window of opportunity where pre-sleep protein supplementation can be an effective nutrient timing strategy to optimize skeletal muscle reconditioning.

    October 25, 2016   doi: 10.1152/ajpendo.00325.2016   open full text
  • Differential effects of biphenol A diglicydyl ether on bone quality and marrow adiposity in ovary-intact and ovariectomized rats.
    Li, G., Xu, Z., Hou, L., Li, X., Li, X., Yuan, W., Polat, M., Chang, S.
    AJP Endocrinology and Metabolism. October 18, 2016

    Bisphenol A diglycidyl ether (BADGE), a PPAR2 antagonist, has been shown to inhibit marrow adipogenesis and promotes bone formation in intact animals. We investigated the impact of BADGE on a new and more clinically relevant physiological model, the ovariectomized (OVX) rat model. Forty female Wistar rats were divided into four treatment groups (n=10/group): sham+vehicle, sham+BADGE, OVX+vehicle and OVX+BADGE for 12 weeks. Postmortem analyses included MRI, micro-CT, serological test, histomorphometry, biomechanical tests, RT-PCR and western blot. Overall, OVX induced a sequential marrow fat expansion accompanied by bone deterioration. Compared with OVX controls, BADGE reduced fat fraction of the distal femur by 36.3%, adipocyte density by 33.0%, adipocyte size by 28.6%, adipocyte volume percentage by 57.8%, adipogenic markers PPAR2 and C/EBP by 50% in OVX rats. Similar results were observed in sham rats vs vehicle. BADGE could promote bone quality in sham rats; however, BADGE did not significantly improve trabecular microarchitecture, biomechanical strength and dynamic histomorphometric parameters except for trabecular separation in OVX rats. We concluded that early BADGE treatment at a dose of 30mg/kg attenuates marrow adiposity in ovary-intact and OVX rats, and stimulates bone formation in ovary-intact rats, but does not significantly rescue bone quality in OVX rats.

    October 18, 2016   doi: 10.1152/ajpendo.00267.2016   open full text
  • Divergent Functions of Endotrophin on Different Cell Populations in Adipose Tissue.
    Zhao, Y., Gu, X., Zhang, N., Kolonin, M. G., An, Z., Sun, K.
    AJP Endocrinology and Metabolism. October 11, 2016

    Endotrophin is a cleavage product of collagen 6 (Col6) in adipose tissue (AT). Previously, we demonstrated that endotrophin serves as a costimulator to trigger fibrosis and inflammation within the unhealthy AT milieu. However, how endotrophin affects lipid storage and breakdown in AT and how different cell types in AT respond to endotrophin stimulation remain unknown. In the current study, by using a doxycycline (Dox)-inducible mouse model, we observed significant upregulation of adipogenic genes in the WAT of endotrophin Tg mice. We further showed that the mice exhibited inhibited lipolysis and accelerated hypertrophy and hyperplasia in WAT. To investigate the effects of endotrophin in vitro, we incubated different cell types from AT with conditioned medium from endotrophin-overexpressing 293T cells. We found that endotrophin activated multiple pathological pathways in different cell types. Particularly, in 3T3-L1 adipocytes, endotrophin triggered a fibrotic program by upregulating collagen genes and promoted abnormal lipid accumulation by downregulating hormone-sensitive lipolysis (HSL) gene and decreasing HSL phosphorylation levels. In macrophages isolated from white AT (WAT), endotrophin stimulated higher expression of the collagen linking enzyme lysyl oxidase (LOX) and M1 pro-inflammatory marker genes. In the stromal vascular fraction (SVF) isolated from WAT, endotrophin induced upregulation of both pro-fibrotic and pro-inflammatory genes. In conclusion, our study provides a new perspective on the effect of endotrophin in abnormal lipid accumulation and a mechanistic insight into the roles played by adipocytes and a variety of other cell types in AT in shaping the unhealthy microenvironment upon endotrophin treatment.

    October 11, 2016   doi: 10.1152/ajpendo.00314.2016   open full text
  • Caspase-1 Deficiency Promotes High-Fat Diet-Induced Adipose Tissue Inflammation and the Development of Obesity.
    Kimura, H., Karasawa, T., Usui, F., Kawashima, A., Endo, Y., Kobayashi, M., Sadatomo, A., Nakamura, J., Iwasaki, Y., Yada, T., Tsutsui, H., Kasahara, T., Takahashi, M.
    AJP Endocrinology and Metabolism. October 04, 2016

    Caspase-1 is a cysteine protease responsible for the processing of the proinflammatory cytokine interleukin-1β and activated by the formation of inflammasome complexes. Although several investigations have found a link between diet-induced obesity and caspase-1, the relationship of remains controversial. Here, we found that mice deficient in caspase-1 were susceptible to high-fat diet-induced obesity with increased adiposity as well as normal lipid and glucose metabolism. Caspase-1 deficiency clearly promoted the infiltration of inflammatory macrophages and increased the production of C-C motif chemokine ligand 2 (CCL2) in the adipose tissue. The dominant cellular source of CCL2 was stromal vascular fraction rather than adipocytes in the adipose tissue. These findings demonstrate a critical role of caspase-1 in macrophage-driven inflammation in the adipose tissue and the development of obesity. These data provide novel insights into the mechanisms underlying inflammation in the pathophysiology of obesity.

    October 04, 2016   doi: 10.1152/ajpendo.00174.2016   open full text
  • Prolonged Sitting Negatively Affects the Postprandial Plasma Triglyceride Lowering Effect of Acute Exercise.
    Kim, I.-Y., Park, S., Chou, T.-H., Trombold, J. R., Coyle, E. F.
    AJP Endocrinology and Metabolism. October 04, 2016

    The interaction of prolonged sitting with physical exercise for maintaining health is unclear. We tested the hypothesis that prolonged siting would have a deleterious effect on postprandial plasma lipemia (PPL, postprandial plasma triglycerides) and abolish the ability of an acute exercise bout to attenuate PPL. Seven healthy young men performed three interventions over 5 days (D1-5) in a randomized crossover design with > 1 week between interventions: 1) sitting >14h/d with hypercaloric energy balance (SH), 2) sitting >14h/d with net energy balance (SB), and 3) active walking/standing with net energy balance (WB) and sitting 8.4 h/d. The first high fat tolerance test (HFTT1) was performed on D3 following 2 days of respective interventions. On the evening of D4 subjects ran on a treadmill for 1-h at ~ 67% VO2max, followed by the second HFTT (HFTT2) on D5. Two days of prolonged sitting increased TG AUCI (i.e., incremental area under the curve for TG), irrespective of energy balance, compared to WB (27% in SH, p=0.003 and 26% in SB, p=0.046). Surprisingly, after four days of prolonged sitting (i.e.; SH and SB), the acute exercise on D4 failed to attenuate TG AUCI or increase relative fat oxidation in HFTT2, compared to HFTT1, independent of energy balance. In conclusion, prolonged sitting over several days was sufficient to amplify PPL and to abolish the beneficial effect of acute exercise on lowering PPL and raising fat oxidation, regardless of energy balance. This underscores the importance of limiting sitting time even in people who have exercised.

    October 04, 2016   doi: 10.1152/ajpendo.00287.2016   open full text
  • Protein Coingestion with Alcohol Following Strenuous Exercise Attenuates Alcohol-Induced Intramyocellular Apoptosis and Inhibition of Autophagy.
    Smiles, W. J., Parr, E. B., Coffey, V. G., Lacham-Kaplan, O., Hawley, J. A., Camera, D. M.
    AJP Endocrinology and Metabolism. September 27, 2016

    Alcohol ingestion decreases post-exercise rates of muscle protein synthesis, but the mechanism(s) (e.g., increased protein breakdown) underlying this observation are unknown. Autophagy is an intracellular "recycling" system required for homeostatic substrate and organelle turnover; its dysregulation may provoke apoptosis and lead to muscle atrophy. We investigated the acute effects of alcohol ingestion on autophagic cell signaling responses to a bout of concurrent (combined resistance- and endurance-based) exercise. In a randomized cross-over design, 8 physically active males completed three experimental trials of concurrent exercise with either post-exercise ingestion of alcohol and carbohydrate (12±2 standard drinks; ALC-CHO), energy-matched alcohol and protein (ALC-PRO), or protein (PRO) only. Muscle biopsies were taken at rest and 2 and 8 h post-exercise. Select autophagy-related gene (Atg) proteins decreased compared to rest with ALC-CHO (P<0.05), but not ALC-PRO. There were parallel increases (P<0.05) in p62 and PINK1, commensurate with a reduction in BNIP3 content, indicating a diminished capacity for mitochondria-specific autophagy (mitophagy) when alcohol and carbohydrate were coingested. DNA fragmentation increased in both alcohol conditions (P<0.05); however, nuclear AIF accumulation preceded this apoptotic response with ALC-CHO only (P<0.05). In contrast, increases in the nuclear content of p53, TFEB and PGC-1α in ALC-PRO were accompanied by markers of mitochondrial biogenesis at the transcriptional (Tfam, SCO2, NRF-1) and translational (COXIV, ATPAF1, VDAC1) level (P<0.05). We conclude that alcohol ingestion following exercise triggers apoptosis, whereas the anabolic properties of protein coingestion may stimulate mitochondrial biogenesis to protect cellular homeostasis.

    September 27, 2016   doi: 10.1152/ajpendo.00303.2016   open full text
  • Inflammation Mediates the Association between Visceral Adiposity and Obstructive Sleep Apnea in Adolescents.
    Gaines, J., Vgontzas, A. N., Fernandez-Mendoza, J., Calhoun, S. L., He, F., Liao, D., Sawyer, M. D., Bixler, E. O.
    AJP Endocrinology and Metabolism. September 20, 2016

    Only a handful of studies, primarily in clinical samples, have reported an association between obesity, inflammation, and obstructive sleep apnea (OSA) in children and adolescents. No studies, however, have examined the pathogenetic link between visceral adiposity, systemic inflammation, and incident OSA in a large general population sample using objective measures of sleep and body fat. Adolescents (n=392; mean age 17.0±2.2y, 54.0% male) from the Penn State Child Cohort (PSCC) underwent 9-hour overnight polysomnography; a DXA scan to assess body fat distribution; and a single fasting blood draw for the assessment of plasma interleukin-6 (IL-6), IL-6 soluble receptor (IL-6 sR), tumor necrosis factor alpha (TNFα), tumor necrosis factor receptor 1A (TNFR1), C-reactive protein (CRP), leptin, and adiponectin levels via ELISA. Visceral fat area was significantly elevated in moderate OSA (AHI ≥ 5), especially in boys. IL-6, CRP, and leptin were highest in adolescents with moderate OSA, even after adjusting for BMI percentile. Mediation analysis revealed that 42% of the association between visceral fat and OSA in adolescents was mediated by IL-6 (p = 0.03), while 82% of the association was mediated by CRP (p = 0.01). These data are consistent with the model of a feed-forward, vicious cycle, in which the release of proinflammatory cytokines by visceral adipocytes largely explains the association between central obesity and OSA; in turn, inflammation is also elevated in OSA independent of BMI. These findings, in a large, representative, non-clinical sample of young people, add to our understanding of the developmental pathogenesis of sleep apnea.

    September 20, 2016   doi: 10.1152/ajpendo.00249.2016   open full text
  • Leucine supplementation stimulates protein synthesis and reduces degradation signal activation in muscle of newborn pigs during acute endotoxemia.
    Hernandez-Garcia, A. D., Columbus, D. A., Manjarin, R., Nguyen, H. V., Suryawan, A., Orellana, R. A., Davis, T. A.
    AJP Endocrinology and Metabolism. September 13, 2016

    Sepsis disrupts skeletal muscle proteostasis and mitigates the anabolic response to leucine (Leu) in muscle of mature animals. We have shown that Leu stimulates muscle protein synthesis (PS) in healthy neonatal piglets. To determine if supplemental Leu can stimulate PS and reduce protein degradation (PD) signaling in neonatal muscle during endotoxemia, overnight fasted neonatal pigs were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic-substrate clamps. Leu or saline was infused during the last hour. Markers of PS and PD were determined in skeletal muscle. Compared to controls, Leu increased PS in longissimus dorsi (LD), gastrocnemius, and soleus muscles. LPS decreased PS in these 3 muscles by 36%, 28%, and 38% but Leu antagonized that reduction by increasing PS by 84%, 81%, and 83%, respectively, when supplemented to LPS. Leu increased eIF3b-raptor interaction, 4EBP1 and S6K1 phosphorylation, and eIF4E·eIF4G complex formation in LD, gastrocnemius, and soleus of control and LPS treated pigs. In LD, LPS increased LC3-II to LC3 ratio and MuRF-1 abundance, but not Atrogin-1 abundance or AMPKα phosphorylation. Leucine supplementation to LPS treated pigs reduced LC3-II/LC3 ratio, MuRF-1 abundance, and AMPKα phosphorylation when compared to LPS alone. In conclusion, parenteral Leu supplementation attenuates the LPS-induced reduction in PS by stimulating mTORC1-dependent translation and may reduce PD by attenuating autophagy-lysosome and MuRF-1 signaling in neonatal skeletal muscle.

    September 13, 2016   doi: 10.1152/ajpendo.00217.2016   open full text
  • Zinc finger protein 407 overexpression upregulates PPAR-target gene expression and improves glucose homeostasis in mice.
    Charrier, A., Wang, L., Stephenson, E. J., Ghanta, S. V., Ko, C.-w., Croniger, C. M., Bridges, D., Buchner, D. A.
    AJP Endocrinology and Metabolism. September 13, 2016

    The peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors are central to the pathophysiology and treatment of metabolic disease through their ability to regulate the expression of genes involved in glucose homeostasis, adipogenesis, and lipid metabolism. However, the mechanism by which PPAR is regulated remains incompletely understood. We generated a transgenic mouse strain (ZFP-TG) that overexpressed Zfp407 primarily in muscle and heart. Transcriptome analysis by RNA-Seq identified 1,300 differentially expressed genes in the muscle of ZFP-TG mice, among which PPAR target genes were significantly enriched. Among the physiologically important PPAR target genes, Glucose transporter (Glut)-4 mRNA and protein levels were increased in heart and muscle. The increase in Glut4 and other transcriptional effects of Zfp407 overexpression together decreased body weight and lowered plasma glucose, insulin, and HOMA-IR scores relative to control littermates. When placed on high fat diet, ZFP-TG mice remained more glucose tolerant than their wild-type counterparts. Cell-based assays demonstrated that Zfp407 synergistically increased the transcriptional activity of all PPAR subtypes, PPARα, PPAR, and PPAR. The increased PPAR activity was not associated with increased PPAR mRNA or protein levels, suggesting that Zfp407 post-translationally regulates PPAR activity. Collectively, these results demonstrate that Zfp407 overexpression improved glucose homeostasis. Thus, Zfp407 represents a new drug target for treating metabolic disease.

    September 13, 2016   doi: 10.1152/ajpendo.00234.2016   open full text
  • Saturated fatty acids activate caspase-4/-5 in human monocytes, triggering IL1{beta} and IL18 release.
    Pillon, N. J., Chan, K. L., Zhang, S., Mejdani, M., Jacobson, M. R., Ducos, A., Bilan, P. J., Niu, W., Klip, A.
    AJP Endocrinology and Metabolism. September 13, 2016

    Obesity is associated with metabolic tissue infiltration by monocyte-derived macrophages. Saturated fatty acids contribute to pro-inflammatory gene induction in tissue-embedded immune cells. However, it is unknown how circulating monocytes -the macrophage precursors- react to high fat environments. In macrophages, saturated fatty acids activate inflammatory pathways and, notably, prime caspase-associated inflammasomes. Inflammasome-activated IL1β contributes to type 2 diabetes. We hypothesized that a) human monocytes from obese patients show caspase activation, and b) fatty acids trigger this response and consequent release of IL1β/IL18. Human peripheral blood monocytes were sorted by flow cytometry and caspase activity was measured with a FLICA dye-based assay. Blood monocytes from obese individuals exhibited elevated caspase activity. To explore the nature and consequence of this activity, human THP1 monocytes were exposed to saturated or unsaturated fatty acids. Caspase activity was revealed by isoform-specific cleavage and enzymatic activity; cytokine expression/release was measured by qPCR and ELISA. Palmitate, but not palmitoleate, increased caspase activity in parallel to the release of IL1β and IL18. Palmitate induced eventual monocyte cell death with features of pyroptosis (an inflammation-linked cell death program involving caspase-4/-5), scored through LDH release, vital dye influx, cell volume changes and nuclear morphology. Notably, selective gene silencing or inhibition of caspase-4/-5 reduced palmitate-induced release of IL1β and IL18. In summary, monocytes from obese individuals present elevated caspase activity. Mechanistically, palmitate activates a pyroptotic program in monocytes through caspase-4/-5 causing inflammatory cytokine release, additional to inflammasomes. These caspases represent potential, novel therapeutic targets to taper obesity-associated inflammation.

    September 13, 2016   doi: 10.1152/ajpendo.00296.2016   open full text
  • Development of a reliable, automated screening system to identify small molecules and biologics that promote human {beta} cell regeneration.
    Aamodt, K. I., Aramandla, R., Brown, J., Fiaschi-Taesch, N., Wang, P., Stewart, A. F., Brissova, M., Powers, A. C.
    AJP Endocrinology and Metabolism. September 13, 2016

    Numerous compounds stimulate rodent β cell proliferation; however, translating these findings to human β cells remains a challenge. To examine human β cell proliferation in response to such compounds, we developed a medium-throughput in vitro method of quantifying adult human β cell proliferation markers. This method is based on high-content imaging of dispersed islet cells seeded in 384-well plates and automated cell counting that identifies fluorescently-labeled β cells with high specificity using both nuclear and cytoplasmic markers. β cells from each donor were assessed for their function and ability to enter the cell cycle by co-transduction with adenoviruses encoding cell cycle regulators cdk6 and cyclin D3. Using this approach, we tested 12 previously identified mitogens including neurotransmitters, hormones, growth factors, and molecules involved in adenosine and Tgf-1β signaling. Each compound was tested in a wide concentration range either in the presence of basal (5 mM) or high (11 mM) glucose. Treatment with control compound harmine, a Dyrk1a inhibitor, led to a significant increase in Ki67+ β cells, while treatment with other compounds had limited to no effect on human β cell proliferation. This new, scalable approach reduces the time and effort required for sensitive and specific evaluation of human β cell proliferation, thus allowing for increased testing of candidate human β cell mitogens.

    September 13, 2016   doi: 10.1152/ajpendo.00515.2015   open full text
  • Healthy Glucocorticoid Receptor N363S Carriers Dysregulate Gene Expression Associated with Metabolic Syndrome.
    Jewell, C. M., Katen, K. S., Barber, L. M., Cannon, C., Garantziotis, S., Cidlowski, J. A.
    AJP Endocrinology and Metabolism. September 06, 2016

    The glucocorticoid receptor single-nucleotide polymorphism (SNP), N363S, has been reported to be associated with metabolic syndrome, Type 2 diabetes and cardiovascular disease. Our aim was to determine how the N363S SNP modifies glucocorticoid receptor signaling in a healthy population of individuals prior to the onset of disease. We examined the function of the N363S SNP in a cohort of subjects from the general population of North Carolina. Eighteen N363S heterozygous carriers and 36 non-carrier, control subjects were examined for clinical and biochemical parameters followed by a low dose dexamethasone suppression test to evaluate glucocorticoid responsiveness. Serum insulin measurements revealed that N363S carriers have higher levels of insulin, although not statistically significant, in comparison to controls. Glucocorticoid receptor protein levels evaluated in peripheral blood mononuclear cells from each clinical subject showed no difference between N363S and control. However, investigation of gene expression profiles in macrophages isolated from controls and N363S carriers using microarray, quantitative RT-PCR and NanoString analyses revealed that the N363S SNP had an altered profile in comparison to control. These changes in gene expression occurred in both the absence and presence of glucocorticoids. Thus, our observed difference in gene regulation between normal N363S SNP carriers and non-carrier controls may underlie the emergence of metabolic syndrome, Type 2 diabetes and cardiovascular disease associated with the N363S polymorphism.-

    September 06, 2016   doi: 10.1152/ajpendo.00105.2016   open full text
  • Aerobic capacity and hepatic mitochondrial lipid oxidation alters susceptibility for chronic high fat diet induced hepatic steatosis.
    Morris, E. M., Meers, G. M. E., Koch, L. G., Britton, S. L., Fletcher, J. A., Shankar, K., Fu, X., Burgess, S. C., Ibdah, J. A., Rector, R. S., Thyfault, J. P.
    AJP Endocrinology and Metabolism. September 06, 2016

    Rats selectively bred for high capacity running (HCR) or low capacity running (LCR) display divergence for intrinsic aerobic capacity and hepatic mitochondrial oxidative capacity, both factors associated with susceptibility for nonalcoholic fatty liver disease (NAFLD). Here we tested if HCR and LCR rats display differences in susceptibility for hepatic steatosis following 16 weeks of high fat diets (HFD) with either 45% or 60% of kcals from fat. The HCR were protected against HFD-induced hepatic steatosis while only the 60%HFD induced steatosis in LCR as marked by a doubling of liver triglycerides. Hepatic complete fatty acid oxidation (FAO) and mitochondrial respiratory capacity were all lower in the LCR compared to HCR. The LCR also displayed lower hepatic complete and incomplete FAO in the presence of etomoxir, suggesting a reduced role for non-carnitine palmitoyltransferase-1 mediated lipid catabolism in LCR vs. HCR. Hepatic complete FAO and mitochondrial respiration were largely unaffected by either chronic HFD; however, 60% HFD feeding markedly reduced 2-pyruvate oxidation, a marker of TCA cycle flux, and mitochondrial complete FAO only in the LCR rats. The LCR displayed lower levels of hepatic long chain acylcarnitines than the HCR but maintained similar levels of hepatic acetyl-carnitine levels, further supporting lower rates of β-oxidation, and TCA cycle flux in the LCR than HCR. Finally, only the LCR displayed early reductions in TCA cycle genes after the acute initiation of a HFD. In conclusion, intrinsically high aerobic capacity confers protection against high fat diet-induced hepatic steatosis through elevated hepatic mitochondrial oxidative capacity.

    September 06, 2016   doi: 10.1152/ajpendo.00178.2016   open full text
  • Leucine Rich Repeat Kinase 1 Regulates Osteoclast Function by Modulating RAC1/Cdc42 Small GTPase Phosphorylation and Activation.
    Zeng, C., Goodluck, H., Qin, X., Liu, B., Mohan, S., Xing, W.
    AJP Endocrinology and Metabolism. September 06, 2016

    Lrrk1 consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphyseal dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1 deficient osteoclasts using pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined if RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and Pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 minutes after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein activated kinase 1 at serine 144, catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function via directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends upon ANK, ROC, WD40, and kinase domains.

    September 06, 2016   doi: 10.1152/ajpendo.00189.2016   open full text
  • 25-hydroxycholesterol impairs endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase.
    Ou, Z.-J., Chen, J., Dai, W.-P., Liu, X., Yang, Y.-K., Li, Y., Lin, Z.-B., Wang, T.-T., Wu, Y.-Y., Su, D.-H., Cheng, T.-P., Wang, Z.-P., Tao, J., Ou, J.-S.
    AJP Endocrinology and Metabolism. September 06, 2016

    Endothelial dysfunction is a key early step in atherosclerosis. 25-hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here we hypothesize that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells (BAECs) were incubated with 25-OHC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS) and AKT as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining, the caspase 3 activity and the expression of Bcl-2, Bax, the cleaved caspase 9 and cleaved caspase 3 were detected by immunoblotting. Finally, aortic ring from SD rats were isolated and treated with 25-OHC and the endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of AKT and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cells apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase 9 and cleaved caspase 3 expressions and the caspase 3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis.

    September 06, 2016   doi: 10.1152/ajpendo.00218.2016   open full text
  • Novel Single Skeletal Muscle Fiber Analysis Reveals a Fiber Type Selective Effect of Acute Exercise on Glucose Uptake.
    Cartee, G. D., Arias, E. B., Yu, C. S., Pataky, M. W.
    AJP Endocrinology and Metabolism. September 06, 2016

    One exercise session can induce subsequently elevated insulin sensitivity that is largely attributable to greater insulin-stimulated glucose uptake by skeletal muscle. Because skeletal muscle is a heterogeneous tissue comprised of diverse fiber types, our primary aim was to determine exercise effects on insulin-independent and insulin-dependent glucose uptake by single fibers of different fiber types. We hypothesized that each fiber type featuring elevated insulin-independent glucose uptake immediately post-exercise (IPEX) would be characterized by increased insulin-dependent glucose uptake at 3.5h post-exercise (3.5hPEX). Rat epitrochlearis muscles were isolated and incubated with [3H]-2-deoxyglucose. Muscles from IPEX and sedentary (SED) controls were incubated without insulin. Muscles from 3.5hPEX and SED controls were incubated ±insulin. Glucose uptake ([3H]-2-deoxyglucose accumulation) and fiber type (myosin heavy chain isoform expression) were determined for single fibers dissected from the muscles. Major new findings included: 1) insulin-independent glucose uptake was increased IPEX in single fibers of each fiber type (I, IIA, IIB, IIBX and IIX); 2) glucose uptake values from insulin-stimulated type I and IIA fibers exceeded the values for the other fiber types; 3) insulin-stimulated glucose uptake for type IIX exceeded IIB fibers; and 4) the 3.5hPEX group versus SED had greater insulin-stimulated glucose uptake in type I, IIA, IIB and IIBX, but not IIX fibers. Insulin-dependent glucose uptake was increased at 3.5hPEX in each fiber type except IIX fibers even though insulin-independent glucose uptake was increased IPEX in all fiber types (including IIX). Single fiber analysis enabled the discovery of this fiber type-related difference for post-exercise, insulin-stimulated glucose uptake.

    September 06, 2016   doi: 10.1152/ajpendo.00289.2016   open full text
  • Molecular Patterns Of Diffuse And Nodular Parathyroid Hyperplasia In Long Term Hemodialysis.
    Tycova, I., Dusilova Sulkova, S., Stepankova, J., Krejcik, Z., Dostalova Merkerova, M., Stranecky, V., Hruba, P., Girmanova, E., Cernoch, M., Lipar, K., Marada, T., Povysil, C., Viklicky, O.
    AJP Endocrinology and Metabolism. September 06, 2016

    Background: Secondary hyperparathyroidism is a well-known complication of end-stage renal disease (ESRD). Both nodular and diffuse parathyroid hyperplasia occur in ESRD patients. However, their distinct molecular mechanisms remain poorly understood. Methods: Parathyroid tissue obtained from ESRD patients who had undergone parathyroidectomy was used for Illumina transcriptome screening and subsequently for discriminatory gene analysis, pathway mapping and gene-annotation enrichment analysis. Results were further validated using RT-qPCR on the independent larger cohort. Results: Microarray screening proved homogeneity of gene transcripts in hemodialysis patients as compared to transplant cohort and primary hyperparathyroidism, therefore further studies were performed in hemodialysis patients only. Enrichment analysis conducted on 485 differentially expressed genes between nodular and diffuse parathyroid hyperplasia revealed highly significant differences in GO terms and the KEGG database in ribosome structure (p=3.70 x 10-18). Next, RT-qPCR validation of the top differently expressed genes from microarray analysis proved higher expression of RAN guanine nucleotide release factor (RANGRF, p<0.001), calcyclin binding protein (CACYBP, p<0.05) and exocyst complex component 8 (EXOC8, p<0.05) and lower expression of peptidylprolyl cis/trans isomerase and NIMA-interacting 1 (PIN1, p<0.01) mRNA in nodular hyperplasia. Multivariate analysis revealed higher RANGRF and lower PIN1 expression along with parathyroid weight to be associated with nodular hyperplasia. Conclusion: Our study suggests the RANGRF transcript which controls the RNA metabolism to be likely involved in pathways associated with the switch to nodular parathyroid growth. This transcript along with PIN1 transcript which influences the PTH secretion may represent new therapeutical targets to cure secondary hyperparathyroidism.

    September 06, 2016   doi: 10.1152/ajpendo.00517.2015   open full text
  • Short-term high-fat feeding induces islet macrophage infiltration and {beta}-cell replication independent of insulin resistance in mice.
    Woodland, D. C., Liu, W., Leong, J., Sears, M. L., Luo, P., Chen, X.
    AJP Endocrinology and Metabolism. August 30, 2016

    Short-term high fat consumption stimulates mouse islet β-cell replication through unknown mechanisms. Resident macrophages (Ms) are capable of secreting various factors involved in islet development and tissue remodeling. We hypothesized that short-term high fat diet (HFD) promotes M-infiltration in pancreatic islets and that Ms serve as regulators of β-cell replication. To test these hypotheses and dissect mechanisms involved in HFD-induced β-cell replication, adult C57BL/6J mice were fed HFD for 7 days, with or without administration of clodronate-containing liposomes, an M-depleting agent. Mouse body- and epididymal fat pad weights, and non-fasting blood glucose and fasting serum insulin levels were measured; and pancreatic islet β-cell replication, oxidative stress, apoptosis, and M infiltration were examined. Short-term HFD promoted an increase in body and epididymal fat pad weights and blood glucose levels, along with an increased fasting serum insulin concentration. β-cell replication, islet M infiltration, and the percentage of iNOS-positive Ms in the islets increased significantly in mice fed HFD. Immunofluorescence staining for 8-OHdG or activated caspase-3 revealed no significant induction of DNA damage or apoptosis, respectively. In addition, no change in stromal derived factor-1 expressing cells was found in the short-term HFD group. Despite continuous elevation of non-fasting blood glucose and fasting serum insulin levels, depletion of Ms through treatments of clodronate abrogated HFD-induced β-cell replication. These findings demonstrated that the HFD-induced M infiltration is responsible for β-cell replication. This study suggests the existence of macrophage-mediated mechanisms in β-cell replication that are independent of insulin-resistance.

    August 30, 2016   doi: 10.1152/ajpendo.00092.2016   open full text
  • Treatment of Non-Alcoholic Fatty Liver Disease (NAFLD): Role of AMPK.
    Smith, B. K., Marcinko, K., Desjardins, E. M., Lally, J. S., Ford, R. J., Steinberg, G. R.
    AJP Endocrinology and Metabolism. August 30, 2016

    Non-alcoholic fatty liver disease (NAFLD) is a growing worldwide epidemic and an important risk factor for the development of insulin resistance, type 2 diabetes, non-alcoholic steatohepatitis (NASH) and hepatic cellular carcinoma (HCC). Despite the prevalence of NAFLD, lifestyle interventions involving exercise and weight loss are the only accepted treatments for this disease. Over the last decade numerous experimental compounds have been shown to improve NAFLD in pre-clinical animal models and many of these therapeutics have been shown to increase the activity of the cellular energy sensor AMP-activated protein kinase (AMPK). As AMPK activity is reduced by inflammation, obesity and diabetes, increasing AMPK activity has been viewed as a viable therapeutic strategy to improve NAFLD. In this review we propose 3 primary mechanisms by which AMPK activation may improve NAFLD. In addition, we examine the mechanisms by which AMPK is activated. Finally, we identify 27 studies that have used AMPK activators to reduce NAFLD. Future considerations for studies examining the relationship between AMPK and NAFLD are highlighted.

    August 30, 2016   doi: 10.1152/ajpendo.00225.2016   open full text
  • A benzimidazole derivative small molecule 991 enhances AMPK activity and glucose uptake induced by AICAR or contraction in skeletal muscle.
    Bultot, L., Jensen, T. E., Lai, Y.-C., Madsen, A. L., Collodet, C., Kviklyte, S., Deak, M., Yavari, A., Foretz, M., Ghaffari, S., Bellahcene, M., Ashrafian, H., Rider, M. H., Richter, E. A., Sakamoto, K.
    AJP Endocrinology and Metabolism. August 30, 2016

    AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α subunit (α1, α2) and regulatory β (β1, β2) and (1, 2, 3) subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently-described potent AMPK-activator called 991, in combination with a commonly used activator AICAR or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that 3 is exclusively expressed in skeletal muscle and is implicated in contraction-induced glucose transport, we measured the activity of AMPK3- as well as ubiquitously expressed 1-containing complexes. We initially validated specificity of the antibodies for the assessment of isoform-specific AMPK activity using respective AMPK-deficient mouse models. We observed that a low dose of 991 (5 μM) stimulated a modest or negligible activity of both 1- and 3-containing AMPK complexes. Dual treatment with 991 and AICAR or 991 and contraction profoundly enhanced AMPK1-/3-complex activation and glucose transport compared to any of the single treatments. The study demonstrates a utility of dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types including skeletal muscle.

    August 30, 2016   doi: 10.1152/ajpendo.00237.2016   open full text
  • Direct Effect of Glucocorticoids on Glucose-Activated Adult Rat Beta Cells Increases their Cell Number and their Functional Mass for Transplantation.
    Assefa, Z., Akbib, S., Lavens, A., Stange, G., Ling, Z., Hellemans, K. H., Pipeleers, D.
    AJP Endocrinology and Metabolism. August 23, 2016

    Compounds that increase beta cell number can serve beta cell replacement therapies in diabetes. In vitro studies have identified several agents that can activate DNA synthesis in primary beta cells, however in small percentages of cells and without demonstration of increases in cell number. We used whole-well multi-parameter imaging to first screen a library of 1280 compounds for their ability to recruit adult rat beta cells into DNA synthesis and then assess influences of stimulatory agents on the number of living cells. The four compounds with highest beta cell recruitment were glucocorticoid-receptor ligands (GC). The GC-effect occurred in glucose-activated beta cells and was associated with increased glucose-utilization and oxidation. Hydrocortisone and methylprednisolone almost doubled the number of beta cells in two weeks. The expanded cell population provided an increased functional beta cell mass for transplantation in diabetic animals. These effects are age-dependent; they did not occur in neonatal rat beta cells where GC-exposure suppressed basal replication and was cytotoxic. It is concluded that glucocorticoids can induce replication of adult rat beta cells through a direct action, with intercellular differences in responsiveness that have been related to differences in glucose-activation and in age. These influences can explain variability in GC-induced activation of DNA synthesis in rat and human beta cells. Our study also demonstrated that beta cells can be expanded in vitro to increase the size of metabolically adequate grafts.

    August 23, 2016   doi: 10.1152/ajpendo.00070.2016   open full text
  • Loss of CTRP1 disrupts glucose and lipid homeostasis.
    Rodriguez, S., Lei, X., Petersen, P. S., Tan, S. Y., Little, H. C., Wong, G. W.
    AJP Endocrinology and Metabolism. August 23, 2016

    C1q/TNF-related protein 1 (CTRP1) is a conserved plasma protein of the C1q family with notable metabolic and cardiovascular functions. We previously showed that CTRP1 infusion lowers blood glucose and that transgenic mice with elevated circulating CTRP1 are protected from diet-induced obesity and insulin resistance. Here, we use a genetic loss-of-function mouse model to address the requirement of CTRP1 for metabolic homeostasis. Despite similar body weight, food intake, and energy expenditure, Ctrp1 knockout (KO) mice fed a low-fat diet (LFD) developed insulin resistance and hepatic steatosis. Impaired glucose metabolism in Ctrp1-KO mice was associated with increased hepatic gluconeogenic gene expression and decreased skeletal muscle glucose transporter GLUT4 levels and AMPK activation. Loss of CTRP1 enhanced clearance of orally-administered lipids but did not affect intestinal lipid absorption, hepatic VLDL-triglyceride export, or lipoprotein lipase activity. In contrast to triglycerides, hepatic cholesterol levels were reduced in Ctrp1-KO mice, paralleling the reduced expression of cholesterol synthesis genes. Contrary to expectations, when challenged with a high-fat diet (HFD) to induce obesity, Ctrp1-KO mice had increased physical activity and reduced body weight, adiposity, and expression of lipid synthesis and fibrotic genes in adipose tissue; these phenotypes were linked to elevated FGF-21 levels. Due in part to increased hepatic AMPK activation and reduced expression of lipid synthesis genes, Ctrp1-KO mice fed a HFD also had reduced liver and serum triglyceride and cholesterol levels. Together, these results provide genetic evidence to establish the significance of CTRP1 to systemic energy metabolism in different metabolic and dietary contexts.

    August 23, 2016   doi: 10.1152/ajpendo.00087.2016   open full text
  • Does the muscle protein synthetic response to exercise and amino acid-based nutrition diminish with advancing age? A systematic review.
    Shad, B. J., Thompson, J. L., Breen, L.
    AJP Endocrinology and Metabolism. August 23, 2016

    Our aim was to assess whether the muscle protein synthesis (MPS) response to acute exercise and/or amino acid-based nutrition is attenuated in older compared with young individuals. A systematic review was conducted on studies that directly examined the influence of age on the MPS response to exercise and/or amino acid-based nutrition. Each study arm was synthesised and reported as providing sufficient or insufficient 'evidence of age-related muscle anabolic resistance'. Subsequently, three models were established to compare age-related differences in the MPS response to different anabolic stimuli. Following exercise alone, 8 of the 17 study arms provided sufficient 'evidence of age-related muscle anabolic resistance'. In response to amino acid-based nutrition, 8 of the 21 study arms provided sufficient 'evidence of age-related muscle anabolic resistance'. When exercise and amino acid-based nutrition were combined, only 2 of the 10 study arms provided sufficient 'evidence of age-related muscle anabolic resistance'. Our results highlight that optimisation of exercise and amino acid-based nutrition is sufficient to overcome age-related muscle anabolic resistance. However, the exercise volume and/or the amino acid/protein dose and leucine content must exceed a certain threshold to stimulate equivalent MPS rates in young and older adults, below which age-related muscle anabolic resistance may manifest.

    August 23, 2016   doi: 10.1152/ajpendo.00213.2016   open full text
  • Insulin does not Stimulate Muscle Protein Synthesis during Increased Plasma Branched-chain Amino Acids Alone but Decreases Whole-body Proteolysis in Humans.
    Everman, S., Meyer, C., Tran, L., Hoffman, N., Carroll, C. C., Dedmon, W. L., Katsanos, C. S.
    AJP Endocrinology and Metabolism. August 16, 2016

    Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primarily role in stimulating muscle protein synthesis, and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy, young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. Half of the subjects were studied in the presence of postabsorptive BCAA concentrations (Control), while the other half in the presence of increased plasma BCAA (BCAA). When compared to that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/hour) did not change (P > 0 .05) during insulin in either the Control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs 0.05 ± 0.01) experiments. Insulin decreased (P < 0.01) whole-body phenylalanine rate of appearance (umol/kg/min), indicating suppression of muscle proteolysis, both in the Control (1.02 ± 0.04 vs 0.76 ± 0.04) and the BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA.

    August 16, 2016   doi: 10.1152/ajpendo.00120.2016   open full text
  • Central GIP signaling stimulates peripheral GIP release and promotes insulin and pancreatic polypeptide secretion in nonhuman primates.
    Higgins, P. B., Shade, R. E., Rodriguez-Sanchez, I. P., Garcia-Forey, M., Tejero-Barrera, M. E., Voruganti, V. S., Cole, S. A., Comuzzie, A. G., Folli, F.
    AJP Endocrinology and Metabolism. August 16, 2016

    Glucose-dependent insulinotropic polypeptide (GIP) has important actions on whole body metabolic function. GIP and its receptor are also present in the central nervous system and have been linked to neurotrophic actions. Metabolic effects of central nervous system GIP signaling have not been reported. We investigated whether centrally administered GIP could increase peripheral plasma GIP concentrations and influence the metabolic response to a mixed macronutrient meal in nonhuman primates. An infusion and sampling system was developed to enable continuous intracerebroventricular (ICV) infusions with serial venous sampling in conscious nonhuman primates. Male baboons (Papio Sp.) that were healthy and had normal body weights (28.9 ±2.1Kg) were studied (n=3). Animals were randomized to receive continuous intracerebroventricular (ICV) infusions of GIP ( or vehicle prior to and over the course of a 300min. mixed meal test (15kcal/kg, 1.5g glucose/kg) on two occasions. A significant increase in plasma GIP concentration was observed under ICV GIP infusion (66.5 ±8.0 vs. 680.6 ±412.8pg/mL, p=0.04), before administration of the mixed meal. Increases in postprandial, but not fasted, insulin (p=0.01) and pancreatic polypeptide (p=0.04) were also observed under ICV GIP. Effects of ICV GIP on fasted or postprandial glucagon, glucose, triglyceride, and free fatty acids were not observed. Our data demonstrate that central GIP signaling can promote increased plasma GIP concentrations independent of nutrient stimulation and increase insulin and pancreatic polypeptide responses to a mixed meal.

    August 16, 2016   doi: 10.1152/ajpendo.00166.2016   open full text
  • Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase 1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.
    Vavrova, E., Lenoir, V., Alves-Guerra, M.-C., Denis, R. G., Castel, J., Esnous, C., Dyck, J. R. B., Luquet, S., Metzger, D., Bouillaud, F., Prip-Buus, C.
    AJP Endocrinology and Metabolism. August 09, 2016

    Impaired skeletal muscle mitochondrial fatty acid oxidation (mFAO) has been implicated in the etiology of insulin resistance. Carnitine palmitoyltransferase 1 (CPT1) is a key regulatory enzyme of mFAO whose activity is inhibited by malonyl-CoA, a lipogenic intermediate. Whereas increasing CPT1 activity in vitro has been shown to exert a protective effect against lipid-induced insulin resistance in skeletal muscle cells, only few studies have addressed this issue in vivo. We thus examined whether a direct modulation of muscle CPT1/malonyl-CoA partnership is detrimental or beneficial for insulin sensitivity in the context of diet-induced obesity. By using a Cre-LoxP recombination approach, we generated mice with skeletal muscle-specific and inducible expression of a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl-CoA inhibition. When fed control chow, homozygous CPT1mt transgenic (dbTg) mice exhibited decreased CPT1 sensitivity to malonyl-CoA inhibition in isolated muscle mitochondria, which was sufficient to substantially increase ex-vivo muscle mFAO capacity and whole-body FA utilization in vivo. Moreover, dbTg mice were less prone to high-fat/high-sucrose (HFHS) diet-induced insulin resistance and muscle lipotoxicity despite similar body weight gain, adiposity, and muscle malonyl-CoA content. Interestingly, these CPT1mt protective effects in dbTg-HFHS mice were associated with preserved muscle insulin signaling, increased muscle glycogen content and up-regulation of key genes involved in muscle glucose metabolism. These beneficial effects of muscle CPT1mt expression suggest that a direct modulation of the malonyl-CoA/CPT1 partnership in skeletal muscle could represent a potential strategy to prevent obesity-induced insulin resistance.

    August 09, 2016   doi: 10.1152/ajpendo.00020.2016   open full text
  • Hepatic Functions of GLP-1 and Its Based Drugs: Current Disputes and Perspectives.
    Jin, T., Weng, J.
    AJP Endocrinology and Metabolism. August 09, 2016

    Although GLP-1 analogues were created as therapeutic incretins; extra-pancreatic functions of these drugs, as well as native GLP-1, have been broadly recognized. Among them, the hepatic functions are particularly important. Postprandial GLP-1 release contributes to insulin secretion, which represses hepatic glucose production. This indirect effect of GLP-1 is known as the gut-pancreas-liver axis. Great efforts have been made to determine whether GLP-1 and its analogues possess direct metabolic effects on the liver, as the determination of the existence of direct hepatic effects may advance the therapeutic theory and clinical practice on subjects with insulin resistance. Furthermore, recent investigations on the metabolic beneficial effects of previously assumed "degradation" products of GLP-1, including GLP-128-36 and GLP-132-36, in the liver and elsewhere have drawn intensive attention. Such investigations may further improve the development and the usage of GLP-1 based drugs. Here we have reviewed the current advancement and the existing controversies on the exploration of direct hepatic functions of GLP-1, and presented our perspectives that the direct hepatic metabolic effects of GLP-1 could be a GLP-1 receptor independent event involving Wnt signaling pathway activation.

    August 09, 2016   doi: 10.1152/ajpendo.00069.2016   open full text
  • Connective tissue growth factor (Ctgf) is critical for proper {beta}-cell function and pregnancy-induced {beta}-cell hyperplasia in adult mice.
    Pasek, R. C., Dunn, J. C., Elsakr, J. M., Aramandla, M., Matta, A. R., Gannon, M.
    AJP Endocrinology and Metabolism. July 26, 2016

    During pregnancy, maternal β-cells undergo compensatory changes including increased β-cell mass and enhanced glucose-stimulated insulin secretion. Failure of these adaptations to occur results in gestational diabetes mellitus. The secreted protein, Connective tissue growth factor (Ctgf), is critical for normal β-cell development and promotes regeneration after partial β-cell ablation. During embryogenesis, Ctgf is expressed in pancreatic ducts, vasculature, and β-cells. In adult pancreas, Ctgf is expressed only in the vasculature. Here we show that pregnant mice with global Ctgf haploinsufficiency (CtgfLacZ/+) have an impairment in maternal β-cell proliferation; no difference was observed in virgin CtgfLacZ/+ females. Using a conditional Ctgf allele, we found that mice with a specific inactivation of Ctgf in endocrine cells (CtgfEndo) develop gestational diabetes during pregnancy, but this is due to a reduction in glucose-stimulated insulin secretion rather than impaired maternal β-cell proliferation. Moreover, virgin CtgfEndo females also display impaired GSIS with glucose intolerance, indicating that underlying β-cell dysfunction precedes the development of gestational diabetes in this animal model. This is the first time a role for Ctgf in β-cell function has been reported.

    July 26, 2016   doi: 10.1152/ajpendo.00194.2016   open full text
  • Cortical bone loss caused by glucocorticoid excess requires RANKL production by osteocytes and is associated with reduced OPG expression in mice.
    Piemontese, M., Xiong, J., Fujiwara, Y., Thostenson, J. D., O'Brien, C. A.
    AJP Endocrinology and Metabolism. July 26, 2016

    Glucocorticoid excess is a major cause of low bone mass and fractures. Glucocorticoid administration decreases cortical thickness and increases cortical porosity in mice and these changes are associated with increased osteoclast number at the endocortical surface. Receptor activator of NFkB ligand (RANKL) produced by osteocytes is required for osteoclast formation in cancellous bone as well as the increase in cortical bone resorption caused by mechanical unloading or dietary calcium deficiency. However, whether osteocyte-derived RANKL also participates in the increase in bone resorption caused by glucocorticoid excess is unknown. To address this question, we examined the effects of prednisolone on cortical bone of mice lacking RANKL production in osteocytes. Prednisolone administration increased osteoclast number at the endocortical surface, increased cortical porosity, and reduced cortical thickness in control mice but none of these effects occurred in mice lacking RANKL in osteocytes. Prednisolone administration did not alter RANKL mRNA abundance, but did reduce osteoprotegerin (OPG) mRNA abundance, in osteocyte-enriched cortical bone. Similarly, dexamethasone suppressed OPG, but not RANKL, production in cortical bone organ cultures and primary osteoblasts. These results demonstrate that RANKL produced by osteocytes is required for the cortical bone loss caused by glucocorticoid excess but suggest that the changes in endocortical resorption are driven by reduced OPG rather than elevated RANKL expression.

    July 26, 2016   doi: 10.1152/ajpendo.00219.2016   open full text
  • Irisin exerts dual effects on browning and adipogenesis of human white adipocytes.
    Zhang, Y., Xie, C., Wang, H., Foss, R., Clare, M., George, E. V., Li, S., Katz, A., Cheng, H., Ding, Y., Tang, D., Reeves, W. H., Yang, L.-J.
    AJP Endocrinology and Metabolism. July 19, 2016

    Objectives: To better understand the role of irisin in humans, we examined the effects of irisin in human primary adipocytes and fresh human subcutaneous white adipose tissue (scWAT). Methods: Human primary adipocytes derived from 25 female donors' fresh scWAT were used to examine the effects of irisin on browning and mitochondrial respiration and preadipocytes were used to examine the effects of irisin on adipogenesis and osteogenesis. Cultured fragments of scWAT were used for investigating signal transduction pathways as well as uncoupling protein 1 (UCP1). Individual responses to irisin in scWAT were correlated with basal expression levels of brown/beige genes. Results: Irisin up-regulated the expression of browning-associated genes and UCP1 protein in both cultured primary mature adipocytes and fresh adipose tissues. It also significantly increased thermogenesis at 5 nM by elevating cellular energy metabolism (OCR & ECAR).Treating human scWAT with irisin increased UCP1 expression by activating the ERK and P38 MAPK signaling. Blocking either pathway with specific inhibitors abolished irisin-induced UCP1 upregulation. In contrast, UCP1 in human brown adipose tissue (BAT) was insensitive to irisin. Basal levels of brown/beige and FNDC5 genes correlated positively with the browning response of scWAT to irisin. In addition, irisin significantly inhibited adipogenic differentiation, but promoted osteogenic differentiation. Conclusions: Irisin promotes "browning" of mature adipocytes and scWAT and increases cellular thermogenesis of adipocytes, but inhibits adipogenesis and promotes osteogenesis during lineage-specific differentiation. Our findings provide valuable insights into for further exploring the therapeutic use of irisin in obesity and exercise-associated bone formation.

    July 19, 2016   doi: 10.1152/ajpendo.00094.2016   open full text
  • Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in Type-I Diabetic humans and mice.
    Robinson, M. M., Dasari, S., Karakelides, H., Bergen, H. R., Nair, K. S.
    AJP Endocrinology and Metabolism. July 19, 2016

    Insulin regulates skeletal muscle protein degradation but the types of proteins being degraded in vivo remains to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as Low Molecular Weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin treated streptozotocin (STZ) diabetic mice for 6 and 96 hours and for 8 hours in Type-I Diabetic humans (T1D) for comparison to insulin treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed ~80% of proteins were consistently detected. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation including those involved in mitochondrial function, proteome homeostasis, nDNA support and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit (+0.5 fold, p=0.007) and cytochrome c oxidase subunit 6 (+0.305 fold, p=0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increased degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis especially of mitochondria.

    July 19, 2016   doi: 10.1152/ajpendo.00175.2016   open full text
  • Liraglutide Prevents Microvascular Insulin Resistance and Preserves Muscle Capillary Density in High Fat Diet Fed Rats.
    Chai, W., Fu, Z., Aylor, K., Barrett, E. J., Liu, Z.
    AJP Endocrinology and Metabolism. July 19, 2016

    Muscle microvasculature critically regulates endothelial exchange surface area to facilitate trans-endothelial delivery of insulin, nutrients and oxygen to myocytes. Insulin resistance blunts insulin-mediated microvascular recruitment and decreases muscle capillary density; both contribute to lower microvascular blood volume. Glucagon-like peptide 1 (GLP-1) and its analogs are able to dilate blood vessels and stimulate endothelial cell proliferation. In this study, we aim to determine the effects of sustained stimulation of the GLP-1 receptors on insulin-mediated capillary recruitment and metabolic insulin responses, small arterial endothelial function and muscle capillary density. Rats were fed a high fat diet (HFD) for 4 weeks with or without simultaneous administration of liraglutide and subject to a euglycemic hyperinsulinemic clamp for 120 min after an overnight fast. Insulin-mediated muscle microvascular recruitment and muscle oxygenation were determined before and during insulin infusion. Muscle capillary density was determined and distal saphenous artery was used for determination of endothelial function and insulin-mediated vasodilation. HFD induced muscle microvascular insulin resistance and small arterial vessel endothelial dysfunction and decreased muscle capillary density. Simultaneous treatment of HFD fed rats with liraglutide prevented all these changes and improved insulin-stimulated glucose disposal. These were associated with a significantly increased AMPK phosphorylation and the expressions of VEGF and its receptors. We conclude that GLP-1 receptor agonists may exert its salutary glycemic effect via improving microvascular insulin sensitivity and muscle capillary density during the development of insulin resistance and early use of GLP-1R agonists may attenuate metabolic insulin resistance as well as prevent cardiovascular complications of diabetes.

    July 19, 2016   doi: 10.1152/ajpendo.00205.2016   open full text
  • Liver glycogen metabolism during and after prolonged endurance-type exercise.
    Gonzalez, J. T., Fuchs, C. J., Betts, J. A., van Loon, L. J. C.
    AJP Endocrinology and Metabolism. July 19, 2016

    Carbohydrate and fat are the main substrates utilized during prolonged endurance-type exercise. The relative contribution of each is primarily determined by the intensity and duration of exercise, along with individual training and nutritional status. During moderate-to-high intensity exercise, carbohydrate represents the main substrate source. As endogenous carbohydrate stores (primarily in liver and muscle) are relatively small, endurance-type exercise performance/capacity is often limited by endogenous carbohydrate availability. Much exercise metabolism research to date has focused on muscle glycogen utilization with little attention to the contribution of liver glycogen. 13C magnetic resonance spectroscopy permits direct, non-invasive measurements of liver glycogen content and has increased understanding of the relevance of liver glycogen during exercise. In contrast to muscle, endurance-trained athletes do not exhibit elevated basal liver glycogen concentrations. However, there is evidence that liver glycogenolysis may be lower in endurance-trained athletes compared to untrained controls during moderate-to-high intensity exercise. Liver glycogen sparing in an endurance-trained state may therefore partly account for training-induced performance/capacity adaptations during prolonged (>90 min) exercise. Ingestion of carbohydrate at a relatively high rate (>1.5 g/min) can prevent liver glycogen depletion during moderate-intensity exercise, independent of the type of carbohydrate (e.g. glucose vs sucrose) ingested. To minimize gastrointestinal discomfort, it is recommended to ingest specific combinations or types of carbohydrates (glucose plus fructose and/or sucrose). By co-ingesting glucose with either galactose or fructose, post-exercise liver glycogen repletion rates can be doubled. There are currently no guidelines for carbohydrate ingestion to maximize liver glycogen repletion.

    July 19, 2016   doi: 10.1152/ajpendo.00232.2016   open full text
  • Imeglimin Lowers Glucose Primarily by Amplifying Glucose-Stimulated Insulin Secretion in High Fat Fed Rodents.
    Perry, R. J., Cardone, R. L., Petersen, M. C., Zhang, D., Fouqueray, P., Hallakou-Bozec, S., Bolze, S., Shulman, G. I., Petersen, K. F., Kibbey, R. G.
    AJP Endocrinology and Metabolism. July 12, 2016

    Imeglimin is a promising new oral antihyperglycemic agent, which has been studied in clinical trials as a possible mono- or add-on therapy to lower fasting plasma glucose and improve hemoglobin A1c (1-3, 7). Imeglimin was shown to improve both fasting and postprandial glycemia and to increase insulin secretion in response to glucose during a hyperglycemic clamp after 1-week treatment in type 2 diabetic patients. However, whether the β-cell stimulatory effect of imeglimin is solely or partially responsible for its effects on glycemia remains to be fully confirmed. Here we show that imeglimin directly activates β-cell insulin secretion in awake rodents without affecting hepatic insulin sensitivity, body composition, or energy expenditure. These data identify a primary amplification rather than triggering β-cell mechanism that explains the acute, antidiabetic activity of imeglimin.

    July 12, 2016   doi: 10.1152/ajpendo.00009.2016   open full text
  • Pregnancy-induced changes in the circadian expression of hepatic clock genes: implications for maternal glucose homeostasis.
    Wharfe, M. D., Wyrwoll, C. S., Waddell, B. J., Mark, P. J.
    AJP Endocrinology and Metabolism. July 12, 2016

    Adaptations in maternal carbohydrate metabolism are particularly important in pregnancy because glucose is the principal energy substrate used by the fetus. As metabolic homeostasis is intricately linked to the circadian system via the rhythmic expression of clock genes, it is likely that metabolic adaptations during pregnancy also involve shifts in maternal circadian function. We hypothesized that maternal adaptation in pregnancy involves changes in the hepatic expression of clock genes, which drive downstream shifts in circadian expression of glucoregulatory genes. Maternal liver and plasma (n = 6-8/group) were collected across 24-h periods (0800, 1200, 1600, 2000, 0000, 0400 h) from C57Bl/6J mice under isoflurane/nitrous oxide anesthesia prior to and on days 6, 10, 14 and 18 of pregnancy (term = day 19). Hepatic expression of clock genes and glucoregulatory genes were determined by RT-qPCR. Hepatic clock gene expression was substantially altered across pregnancy, most notably in late gestation when the circadian rhythmicity of several clock genes was attenuated (up to 64% reduced amplitude on day 18). These changes were associated with a similar decline in rhythmicity of the key glucoregulatory genes Pck1, G6Pase and Gk, and by day 18 Pck1 was no longer rhythmic. Overall, our data show marked adaptations in the liver clock during mouse pregnancy, changes that may contribute to the altered circadian variation in glucoregulatory genes near term. We propose that the observed reduction of daily oscillations in glucose metabolism ensure a sustained supply of glucose to meet the high demands of fetal growth.

    July 12, 2016   doi: 10.1152/ajpendo.00060.2016   open full text
  • Post-Translational Modifications and Dysfunction of Mitochondrial Enzymes in Human Heart Failure.
    Sheeran, F. L., Pepe, S.
    AJP Endocrinology and Metabolism. July 12, 2016

    Deficiency of energy supply is a major complication contributing to the syndrome of heart failure (HF). As the concurrent activity profile of mitochondrial bioenergetic enzymes has not been studied collectively in human HF, our aim was to examine the mitochondrial enzyme defects in left ventricular myocardium obtained from explanted end-stage failing hearts. Compared to non-failing donor hearts, activity rates of complexes I and IV and the Krebs cycle enzymes isocitrate dehydrogenase, malate dehydrogenase and aconitase were lower in HF, as determined spectrophotometrically. However, activity rates of complexes II, III and citrate synthase did not differ significantly between the two groups. Protein expression, determined by western blotting, did not differ between the groups, implying post-translational perturbation. In the face of diminished total glutathione and coenzyme Q10 levels, oxidative modification was explored as an underlying cause of enzyme dysfunction. Of the three oxidative modifications measured, protein carbonylation was significantly increased by 31% in HF (p<0.01; n=18), while levels of 4-hydroxynonenal and protein nitration though elevated, did not differ. Isolation of complexes I, IV and F1FoATP synthase by immunocapture revealed that proteins containing iron-sulphur or heme redox centres were targets of oxidative modification. Energy deficiency in end-stage failing human left ventricle involves impaired activity of key electron transport chain and Krebs cycle enzymes, without altered expression of protein levels. Augmented oxidative modification of crucial enzyme subunit structures implicates dysfunction due to diminished capacity for management of mitochondrial reactive oxygen species, thus contributing further to reduced bioenergetics in human HF.

    July 12, 2016   doi: 10.1152/ajpendo.00127.2016   open full text
  • Defining Human Insulin-like Growth Factor 1 Gene Regulation.
    Mukherjee, A., Alzhanov, D., Rotwein, P.
    AJP Endocrinology and Metabolism. July 12, 2016

    Growth hormone (GH) plays an essential role in controlling somatic growth and in regulating multiple physiological processes in humans and other species. Insulin-like growth factor 1 (IGF1), a conserved secreted 70-amino acid peptide, is a critical mediator of many of the biological effects of GH. Previous studies have demonstrated that GH rapidly and potently promotes IGF1 gene expression in rodents and in some other mammals through the transcription factor Stat5b, leading to accumulation of IGF1 mRNAs and production of IGF1. Despite this progress, very little is known about how GH or other trophic factors control human IGF1 gene expression, in large part because of the absence of any cellular model systems that robustly express IGF1. Here we have addressed mechanisms of regulation of human IGF1 by GH after generating cell lines in which the IGF1 chromosomal locus has been incorporated into a mouse cell line. Using these cells we find that physiological levels of GH rapidly stimulate human IGF1 gene transcription, and identify several potential transcriptional enhancers in chromatin that bind Stat5b in a GH-regulated way. Each of the putative enhancers also activates a human IGF1 gene promoter in reconstitution experiments in the presence of the GH receptor, Stat5b, and GH. We thus have developed a novel experimental platform that now may be used to determine how human IGF1 gene expression is controlled under different physiological and pathological conditions.

    July 12, 2016   doi: 10.1152/ajpendo.00212.2016   open full text
  • {beta}-cell dedifferentiation, reduced duct cell plasticity and impaired {beta}-cell mass regeneration in middle-aged rats.
    Tellez, N., Vilaseca, M., Marti, Y., Pla, A., Montanya, E.
    AJP Endocrinology and Metabolism. July 12, 2016

    Limitations in β-cell regeneration potential in middle-aged animals could contribute to the increased risk to develop diabetes associated with aging. We investigated β-cell regeneration of middle-aged Wistar rats in response to two different regenerative stimuli: partial pancreatectomy (Px+V) and gastrin administration (Px+G). Pancreatic remnants were analyzed 3 and 14 days after surgery. β-cell mass increased in young animals after Px and was further increased after gastrin treatment. In contrast, β-cell mass did not change after Px and after gastrin treatment in middle-aged rats. β-cell replication and individual β-cell size were similarly increased after Px in young and middle-aged animals, and β-cell apoptosis was not modified. Nuclear immunolocalization of neurog3 or nkx6.1 in regenerative duct cells, markers of duct cell plasticity, was increased in young but not in middle-aged Px rats. The pancreatic progenitor-associated transcription factors neurog3 and sox9 were upregulated in islet β-cells of middle-aged rats and were further increased after Px. The percentage of chromogranin A+/hormone- islet cells were was significantly increased in the pancreases of middle-aged Px rats. In summary, the potential for compensatory β-cell hyperplasia and hypertrophy was retained in middle-aged rats, but β-cell dedifferentiation and impaired duct-cell plasticity limited β-cell regeneration.

    July 12, 2016   doi: 10.1152/ajpendo.00502.2015   open full text
  • Low resting metabolic rate in exercise-associated amenorrhea is not due to a reduced proportion of highly metabolically active tissue compartments.
    Koehler, K., Williams, N. I., Mallinson, R. J., Southmayd, E. A., Allaway, H. C. M., De Souza, M. J.
    AJP Endocrinology and Metabolism. July 05, 2016

    Exercising women with menstrual disturbances frequently display a low resting metabolic rate (RMR) when RMR is expressed relative to body size or lean mass. However, normalizing RMR for body size or lean mass does not account for potential differences in the size of tissue compartments with varying metabolic activities. To explore whether the apparent RMR suppression in women with exercise-associated amenorrhea is a consequence of a lower proportion of highly metabolically active tissue compartments or the result of metabolic adaptations related to energy conservation at the tissue level, RMR and metabolic tissue compartments were compared among exercising women with amenorrhea (AMEN, n=42) and exercising women with eumenorrheic, ovulatory menstrual cycles (OV, n=37). RMR was measured using indirect calorimetry and predicted from the size of metabolic tissue compartments as measured by dual-energy X-ray absorptiometry (DXA). Measured RMR was lower than DXA-predicted RMR in AMEN (1215±31 vs. 1327±18 kcal/d, p<0.001) but not in OV (1284±24 vs. 1252±17, p=0.16), resulting in a lower ratio of measured to DXA-predicted RMR in AMEN (91±2%) vs. OV (103±2%, p<0.001). AMEN displayed proportionally more residual mass (p<0.001) and less adipose tissue (p=0.003) when compared to OV. A lower ratio of measured to DXA-predicted RMR was associated with lower serum total triiodothyronine (=0.38, p<0.001) and leptin (=0.32, p=0.004). Our findings suggest that RMR suppression in this population is not the result of a reduced size of highly metabolically active tissue compartments but due to metabolic and endocrine adaptations at the tissue level that are indicative of energy conservation.

    July 05, 2016   doi: 10.1152/ajpendo.00110.2016   open full text
  • Short-term and long-term effects of submaximal maternal exercise on offspring glucose homeostasis and pancreatic function.
    Quiclet, C., Siti, F., Dubouchaud, H., Vial, G., Berthon, P., Fontaine, E., Batandier, C., Couturier, K.
    AJP Endocrinology and Metabolism. July 05, 2016

    Only few studies explored the effects of maternal exercise during gestation on adult offspring metabolism. We set out to test whether maternal controlled submaximal exercise maintained troughout all gestationnal periods induces persistant metabolic changes in the offspring. We used a model of 15 week-old nulliparous female Wistar rats who exercised (Trained group) before and during gestation at a submaximal intensity or remained sedentary (Control group). At weaning, male offspring from Trained dams showed reduced basal glycemia (119.7±2.4 vs 130.5±4.1 mg.dl-1, P<0.05), pancreas relative weight (3.96±0.18 vs 4.54±0.14 body weight-1, P<0.05) and islet mean area (22822±4036 vs 44669±6761 µm2, P<0.05) compared with pups from Control dams. Additionally, they had better insulin secretory capacity when stimulated by glucose 2.8 mM + arginine 20 mM, compared with offspring from Control dams (+96%, P<0.05). At 7 months of age, offspring from Trained mothers displayed altered glucose tolerance (AUC = 15285±527 vs 11898±988 mg.dl-1*120 min, P<0.05), decreased muscle insulin sensitivity estimated by the phosphorylated-PKB/total PKB ratio (-32%, P<0.05) and tended to have a reduced islet insulin secretory capacity compared with rats from Control dams. These results suggest that submaximal maternal exercise modifies short-term male offspring pancreatic function and appears to have rather negative long-term consequences on sedentary adult offspring glucose handling.

    July 05, 2016   doi: 10.1152/ajpendo.00126.2016   open full text
  • Targeting Ceramide Metabolism in Obesity.
    Abursayn, H., Al Batran, R., Ussher, J. R.
    AJP Endocrinology and Metabolism. July 05, 2016

    Obesity is a major health concern that increases the risk for insulin resistance, type 2 diabetes (T2D), and cardiovascular disease. Thus, an enormous research effort has been invested into understanding how obesity-associated dyslipidemia and obesity-induced alterations in lipid metabolism increase the risk for these diseases. Accordingly, it has been proposed that the accumulation of lipid metabolites in organs such as the liver, skeletal muscle, and heart, is critical to these obesity-induced pathologies. Ceramide is one such lipid metabolite that accumulates in our tissues in response to obesity, and both pharmacological and genetic strategies that reduce tissue ceramide levels yield salutary actions on overall metabolic health. We will review herein why ceramide accumulates in tissues during obesity, how an increase in intracellular ceramide impacts cellular signalling and function, as well as potential mechanisms by which reducing intracellular ceramide levels improves insulin resistance, T2D, atherosclerosis, and heart failure. As a reduction in skeletal muscle ceramide levels is frequently associated with improvements in insulin sensitivity in humans, the beneficial findings reported for reducing ceramides in preclinical studies may have clinical application in humans. Therefore, modulating ceramide metabolism may be a novel exciting target for preventing and/or treating obesity-related diseases.

    July 05, 2016   doi: 10.1152/ajpendo.00133.2016   open full text
  • Control of Skeletal Muscle Atrophy in Response to Disuse: Clinical/Preclinical Contentions and Fallacies of Evidence.
    Atherton, P. J., Greenhaff, P. L., Phillips, S. M., Bodine, S. C., Adams, C. M., Lang, C. H.
    AJP Endocrinology and Metabolism. July 05, 2016

    Muscle wasting resulting wholly or in part from disuse represents a serious medical complication, which when prolonged, can increase morbidity and mortality. Although much knowledge has been gained over the past half century, the underlying etiology by which disuse alters muscle proteostasis remains enigmatic. Multidisciplinary and novel methodologies are needed to fill gaps and overcome barriers to improved patient care. The present review highlights seminal concepts from a symposium at Experimental Biology 2016. These proceedings focus on the: (1) role of insulin resistance in mediating disuse-induced changes in muscle protein synthesis (MPS) and breakdown (MPB), as well as cross-talk between carbohydrate and protein metabolism; (2) the relative importance of MPS/MPB in mediating involuntary muscle loss in humans and animals; (3) interpretative limitations associated with MPS/MPB "markers" e.g. MuRF1/MAFbx mRNA; and finally, (4) how OMIC technologies can be leveraged to identify molecular pathways (e.g. ATF4, p53, p21) mediating disuse atrophy. This perspective deals primarily with "simple atrophy" due to unloading. Nonetheless, it is likely disuse is a pervasive contributor to muscle wasting associated with catabolic disease-related atrophy (i.e. due to associated sedentary behaviour of disease burden). Key knowledge gaps and challenges are identified to stimulate discussion and identify opportunities for translational research. Data from animal and human studies highlight both similarities and differences. Integrated preclinical and clinical research is encouraged to better understand the metabolic and molecular underpinnings and, translational relevance, for disuse atrophy. These approaches are crucial to clinically prevent or reverse muscle atrophy, thereby reestablishing homeostasis and recovery.

    July 05, 2016   doi: 10.1152/ajpendo.00257.2016   open full text
  • Hypermetabolism and Hypercatabolism of Skeletal Muscle Accompany Mitochondrial Stress Following Severe Burn Trauma.
    Ogunbileje, J. O., Porter, C., Herndon, D. N., Chao, T., Abdelrahman, D. R., Papadimitriou, A., Chondronikola, M., Zimmers, T. A., Reidy, P. T., Rasmussen, B. B., Sidossis, L. S.
    AJP Endocrinology and Metabolism. July 05, 2016

    Burn trauma results in prolonged hypermetabolism and skeletal muscle wasting. How hypermetabolism contributes to muscle wasting in burn patients remains unknown. We hypothesized that oxidative stress, cytosolic protein degradation, and mitochondrial stress as a result of hypermetabolism contributes to muscle cachexia post-burn. Patients (n=14) with burns covering >30% of their total body surface area (TBSA) were studied. Controls (n=13) were young healthy adults. We found that burn patients were profoundly hypermetabolic at both the skeletal muscle and systemic levels, indicating increased oxygen consumption by mitochondria. In skeletal muscle of burn patients, concurrent activation of mTORC1 signaling and elevation in the fractional synthetic rate (FSR) paralleled increased levels of proteasomes and elevated fractional breakdown rate (FBR). Burn patients had greater levels of oxidative stress markers, as well as higher expression of mtUPR-related genes and proteins, suggesting that burns increased mitochondrial stress and protein damage. Indeed, upregulation of cyto-protective genes suggest hypermetabolism-induced oxidative stress post-burn. In parallel to mtUPR activation post burns, mitochondrial specific proteases (LONP1 and CLPP), and mitochondrial translocases (TIM23, TIM 17B, and TOM40) were upregulated, suggesting increased mitochondrial protein degradation and transport of pre-protein, respectively. Our data demonstrate that proteolysis occurs in both the cytosolic and mitochondrial compartments of skeletal muscle in severely burned patients. Increased mitochondrial protein turnover may be associated with increased protein damage due to hypermetabolism-induced oxidative stress and activation of mtUPR. Our results suggest a novel role for the mitochondria in burn-induced cachexia.

    July 05, 2016   doi: 10.1152/ajpendo.00535.2015   open full text
  • A High Fat Diet (HFD) Impairs Cooling-Evoked Brown Adipose Tissue (BAT) Activation via a Vagal Afferent Mechanism.
    Madden, C. J., Morrison, S. F.
    AJP Endocrinology and Metabolism. June 28, 2016

    In dramatic contrast to rats on a control diet, rats maintained on a high fat diet (HFD) failed to activate brown adipose tissue (BAT) during cooling, despite robust increases in their BAT activity following direct activation of their BAT sympathetic premotor neurons in the raphe pallidus. Cervical vagotomy or blockade of glutamate receptors in the nucleus of the tractus solitarii (NTS) reversed the HFD-induced inhibition of cold-evoked BAT activity. Thus, a HFD does not prevent rats from mounting a robust, centrally-driven BAT thermogenesis; however, a HFD does alter a vagal afferent input to NTS neurons, thereby preventing the normal activation of BAT thermogenesis to cooling. These results, paralleling the absence of cooling-evoked glucose uptake in the BAT of obese humans, reveal a neural mechanism through which consumption of a HFD contributes to reduced energy expenditure, and thus to weight gain.

    June 28, 2016   doi: 10.1152/ajpendo.00081.2016   open full text
  • Novel insights into development of diabetic bladder disorder provided by metabolomic analysis of the rat non-diabetic and diabetic detrusor and urothelial layer.
    Wang, Y., Deng, G. G., Davies, K. P.
    AJP Endocrinology and Metabolism. June 28, 2016

    There are at present no published studies providing a global overview of changes in bladder metabolism resulting from diabetes. Such studies have the potential to provide mechanistic insight into the development of diabetic bladder disorder (DBD). In the present study we compared the metabolome of detrusor and urothelial layer in a one month streptozotocin-induced rat model of Type I diabetes with non-diabetic controls. Our studies revealed diabetes caused both common and differential changes in the detrusor and urothelial layer's metabolome. Diabetes resulted in similar changes in levels of previously described diabetic markers in both tissues, such as glucose, lactate, 2-hydroxybutyrate, branched-chain amino acid degradation products, bile acids and 1,5-anhydroglucitol, as well as markers of oxidative stress. In detrusor (but not urothelial layer) diabetes caused activation of the pentose-phosphate and polyol pathways, concomitant with a reduction in the TCA cycle and β-oxidation. Changes in detrusor energy generating pathways resulted in an accumulation of sorbitol which, through generation of advanced glycation end products, is likely to play a central role in the development of DBD. In the diabetic urothelial layer there was decreased flux of glucose via glycolysis and changes in lipid metabolism, particularly prostaglandin synthesis, which also potentially contributes to detrusor dysfunction.

    June 28, 2016   doi: 10.1152/ajpendo.00134.2016   open full text
  • Elevated expression of the Sertoli cell androgen receptor disrupts male fertility.
    Hazra, R., Upton, D., Desai, R., Noori, O., Jimenez, M., Handelsman, D. J., Allan, C. M.
    AJP Endocrinology and Metabolism. June 28, 2016

    We recently created a unique gain-of-function mouse model with Sertoli cell-specific transgenic androgen receptor expression (TgSCAR) that showed SCAR activity controls the synchronized postnatal development of somatic Sertoli and Leydig cells, and meiotic-postmeiotic germ cells. Moderate TgSCAR (TgSCARm) expression reduced testis size but had no effect on male fertility. Here, we reveal that higher TgSCAR expression (TgSCARH) causes male infertility. Higher SCAR activity, shown by upregulated AR-dependent transcripts (Rhox5, Spinw1), resulted in smaller adult TgSCARH testes (50% of normal), despite normal or elevated circulating and intratesticular testosterone levels. Unlike fertile TgSCARm males, testes of adult TgSCARH males exhibited focal regions of interstitial hypertrophy featuring immature adult Leydig cells, and higher intratesticular dihydrotestosterone and 5α-androstane 3α, 17β-diol levels which are normally associated with pubertal development. Mature TgSCARH testes also exhibited markedly reduced Sertoli cell numbers (70%), although meiotic and postmeiotic germ cell to Sertoli cell ratios were 2-fold higher than normal, suggesting elevated TgSCAR activity supports excessive spermatogenic development. Concurrent with the higher germ cell load of TgSCARH Sertoli cells were increased levels of apoptotic germ cells in TgSCARH relative to TgSCARm testes. In addition, TgSCARH testes displayed unique morphological degeneration that featured accumulated cellular and spermatozoa clusters in dilated channels of rete testes, consistent with reduced epididymal sperm numbers. Our findings reveal for the first time that excessive Sertoli cell AR activity in mature testes can reach a level that disturbs Sertoli-germ cell homeostasis, impacts focal Leydig cell function, reduces sperm output and disrupts male fertility.

    June 28, 2016   doi: 10.1152/ajpendo.00159.2016   open full text
  • Biological underpinnings of breastfeeding challenges: the role of genetics, diet and environment on lactation physiology.
    Lee, S., Kelleher, S. L.
    AJP Endocrinology and Metabolism. June 28, 2016

    Lactation is a dynamic process that has evolved to produce a complex biological fluid that provides nutritive and non-nutritive factors to the nursing offspring. It has long been assumed that once lactation is successfully initiated, the primary factor regulating milk production is infant demand. Thus most interventions have focused on improving breastfeeding education and early lactation support. However, in addition to infant demand, increasing evidence from studies conducted in experimental animal models, production animals and breastfeeding women suggests that a diverse array of maternal factors may also affect milk production and composition. In this review, we provide an overview of our current understanding of the role of maternal genetics and modifiable factors, such as diet and environmental exposures, on reproductive endocrinology, lactation physiology and the ability to successfully produce milk. In order to identify factors that may affect lactation in women, we highlight some information gleaned from studies in experimental animal models and production animals. Finally, we highlight the gaps in current knowledge and provide commentary on future research opportunities aimed at improving lactation outcomes in breastfeeding women to improve the health of mothers and their infants.

    June 28, 2016   doi: 10.1152/ajpendo.00495.2015   open full text
  • Changes in the expression of the type 2 diabetes-associated gene VPS13C in the {beta} cell are associated with glucose intolerance in humans and mice.
    Mehta, Z. B., Fine, N., Pullen, T. J., Cane, M. C., Hu, M., Chabosseau, P., Meur, G., Velayos-Baeza, A., Monaco, A. P., Marselli, L., Marchetti, P., Rutter, G. A.
    AJP Endocrinology and Metabolism. June 21, 2016

    Single nucleotide polymorphisms (SNPs) close to the VPS13C, C2CD4A and C2CD4B genes on chromosome 15q are associated with impaired fasting glucose and increased risk of type 2 diabetes. eQTL analysis revealed an association between possession of risk (C) alleles at a previously-implicated causal SNP, rs7163757, and lowered VPS13C and C2CD4A levels in islets from female (n=40; p<0.041) but not male subjects. Explored using promoter-reporter assays in β and other cell lines, the risk variant at rs7163757, lowered enhancer activity. Mice deleted for Vps13c selectively in the β cell were generated by crossing animals bearing a floxed allele at exon 1 to mice expressing Cre recombinase under Ins1 promoter control (Ins1Cre). Whilst Vps13cfl/fl::Ins1Cre (βVps13cKO) mice displayed normal weight gain compared to control littermates, deletion of Vps13c had little effect on glucose tolerance. Pancreatic histology revealed no significant change in β cell mass in KO mice versus controls and glucose-stimulated insulin secretion from isolated islets was not altered in vitro between control and βVps13cKO mice. However, a tendency was observed in female null mice for lower insulin levels and β cell function (HOMA-B) in vivo. Furthermore, glucose-stimulated increases in intracellular free Ca2+ were significantly increased in islets from female KO mice, suggesting impaired Ca2+ sensitivity of the secretory machinery. The present data thus provide evidence for a limited role for changes in VPS13C expression in conferring altered disease risk at this locus, particularly in females, and suggest that C2CD4A may also be involved.

    June 21, 2016   doi: 10.1152/ajpendo.00074.2016   open full text
  • Proposed Mechanisms For Oligonucleotide Imt504 Induced Diabetes Reversion In A Mouse Model Of Immunodependent Diabetes.
    Bianchi, M. S., BIanchi, S., Hernando-Insua, A., Martinez, L. M., Lago, N., Libertun, C., Chasseing, N. A., Montaner, A. D., Lux-Lantos, V. A.
    AJP Endocrinology and Metabolism. June 21, 2016

    Type 1 diabetes (T1D) originates from autoimmune β-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cells cloning capacity and reverts toxic diabetes in rats. Here we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg/kg/day, sc) in an immunodependent diabetes model: multiple low dose streptozotocin-injected BALB/c mice (40 mg/kg/day, i.p. for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. Long-term results: IMT504 reduced glycemia, induced β-cell recovery and impaired islet infiltration. Short-term analysis: IMT504 induced early blood glucose decrease, infiltration inhibition, increased β-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression: Preproinsulin-2, Proglucagon, Somatostatin, Nestin, Regenerating gene-1 and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated Platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased Regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression, and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate β-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.

    June 21, 2016   doi: 10.1152/ajpendo.00104.2016   open full text
  • Targeted overexpression of mitochondrial catalase protects against cancer chemotherapy-induced skeletal muscle dysfunction.
    Gilliam, L. A. A., Lark, D. S., Reese, L. R., Torres, M. J., Ryan, T. E., Lin, C.-T., Cathey, B. L., Neufer, P. D.
    AJP Endocrinology and Metabolism. June 21, 2016

    The loss of strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and increases mitochondrial H2O2. We hypothesized the combined effect of cancer and chemotherapy in an immunocompetent breast cancer mouse model (E0771) would compromise skeletal muscle mitochondrial respiratory function, leading to an increase in H2O2 emitting potential and impaired muscle function. Here we demonstrate cancer chemotherapy decreases mitochondrial respiratory capacity supported with complex I (pyruvate/glutamate/malate) and complex II (succinate) substrates. Mitochondrial H2O2 emitting potential was altered in skeletal muscle and global protein oxidation was elevated with cancer chemotherapy. Muscle contractile function was impaired following exposure to cancer chemotherapy. Genetically engineering the overexpression of catalase in mitochondria of muscle attenuated mitochondrial H2O2 emission and protein oxidation, preserving mitochondrial and whole muscle function despite cancer chemotherapy. These findings suggest mitochondrial oxidants as a mediator of cancer chemotherapy-induced skeletal muscle dysfunction.

    June 21, 2016   doi: 10.1152/ajpendo.00540.2015   open full text
  • Accuracy and precision of quantitative 31P-MRS measurements of human skeletal muscle mitochondrial function.
    Layec, G., Gifford, J. R., Trinity, J. D., Hart, C. R., Garten, R. S., Park, S. Y., Le Fur, Y., Jeong, E.-K., Richardson, R. S.
    AJP Endocrinology and Metabolism. June 14, 2016

    While theoretically sound, the accuracy and precision of 31P-magnetic resonance spectroscopy (31P-MRS) approaches to quantitatively estimate mitochondrial capacity are not well documented. Therefore, employing four differing models of respiratory control (linear, kinetic and multi-point adenosine diphosphate (ADP), and phosphorylation potential), this study sought to determine the accuracy and precision of 31P-MRS assessments of peak mitochondrial adenosine-triphosphate (ATP) synthesis rate utilizing directly measured peak respiration (State 3) in permeabilized skeletal muscle fibers. In 23 subjects, of different fitness levels, 31P-MRS during a 24-s maximal isometric knee-extension and high-resolution respirometry in muscle fibers from the vastus lateralis was performed. Although significantly correlated with State 3 respiration (r=0.72), both the linear (45±13 mM.min-1) and phosphorylation potential (47±16mM.min-1) models grossly overestimated the calculated in vitro peak ATP synthesis rate (P<0.05). Of the ADP models, the kinetic model was well correlated with State 3 respiration (r=0.72; P<0.05), but moderately overestimated ATP synthesis rate (P<0.05), while the multi-point model, although being somewhat less well correlated with State 3 respiration (r=0.55; P<0.05), most accurately reflected peak ATP synthesis rate. Of note, the PCr recovery time constant (), a qualitative index of mitochondrial capacity, exhibited the strongest correlation with State 3 respiration (r=0.80; P<0.05). Therefore, this study reveals that each of the 31P-MRS data analyses, including PCr , exhibit precision in terms of mitochondrial capacity. As only the multi-point ADP model did not overstimate the peak skeletal muscle mitochondrial ATP synthesis, the multi-point ADP model is the only quantitative approach to exhibit both accuracy and precision.

    June 14, 2016   doi: 10.1152/ajpendo.00028.2016   open full text
  • The expansion of adult stem/progenitor cells and their marker expression fluctuations are linked with pituitary plastic adaptation during gestation and lactancy.
    Vaca, A. M., Guido, C. B., Sosa, L. d. V., Nicola, J. P., Mukdsi, J., Petiti, J. P., Torres, A. I.
    AJP Endocrinology and Metabolism. June 14, 2016

    Extensive evidence has revealed variations in the number of hormone-producing cells in the pituitary gland, which occur under physiological conditions such as gestation and lactancy. It has been proposed that new hormone producing cells differentiate from stem cells. However, exactly how and when this takes place is not clear. In this work, we used immunoelectron-microscopy to identify adult pituitary stem/progenitor cells (SC/P) localized in the marginal zone (MZ) and additionally, we detected GFRa2, Sox2 and Sox9 positive cells in the adenoparenchyma (AP) by fluorescence microscopy. Then, we evaluated fluctuations of SC/P mRNA and protein level markers in MZ and AP during gestation and lactancy. An up-regulation in stemness markers was shown at term of gestation (AT) in MZ, while, there was more progenitor cell marker at middle of gestation and active lactancy. Concerning committed cell marker, we detected a rise in AP at beginning of lactancy (d1L). We performed a BrdU-uptake analysis in MZ and AP cells. The highest level of BrdU-uptake was observed in MZ AT cells whereas, in AP, this was detected in d1L, followed by a decrease in both the MZ and AP. Finally, we detected double-immunostaining for BrdU-GFRa2 in MZ AT cells and BrdU-Sox9 in the AP d1L cells. Taken together, we hypothesize that the expansion of the SC/P niche took place mainly in MZ from pituitary rats in AT and d1L. These results suggest that the SC niche actively participates in pituitary plasticity during these reproductive states, contributing to the origin of hormone cell populations.

    June 14, 2016   doi: 10.1152/ajpendo.00077.2016   open full text
  • Jejunal administration of glucose enhances acyl ghrelin suppression in obese humans.
    Tamboli, R. A., Sidani, R. M., Garcia, A. E., Antoun, J., Isbell, J. M., Albaugh, V. L., Abumrad, N. N.
    AJP Endocrinology and Metabolism. June 07, 2016

    Ghrelin is a gastric hormone that stimulates hunger and worsens glucose metabolism. Circulating ghrelin is decreased after Roux-en-Y gastric bypass (RYGB) surgery; however, the mechanism(s) underlying this change is unknown. We tested the hypothesis that jejunal nutrient exposure plays a significant role in ghrelin suppression after RYGB. Feeding tubes were placed in the stomach or jejunum in 13 obese subjects to simulate pre-RYGB or post-RYGB glucose exposure to the gastrointestinal (GI) tract, respectively, without the confounding effects of caloric restriction, weight loss, and surgical stress. On separate study days, the plasma glucose curves obtained with either gastric or jejunal administration of glucose were replicated with intravenous (IV) infusions of glucose. These "isoglycemic clamps" enabled us to determine the contribution of the GI tract and post-absorptive plasma glucose to acyl ghrelin suppression. Plasma acyl ghrelin levels were suppressed to a greater degree with jejunal glucose administration compared to gastric glucose administration (P<0.05). Jejunal administration of glucose also resulted in a greater suppression of acyl ghrelin than the corresponding isoglycemic glucose infusion (P≤0.01). However, gastric and isoglycemic IV glucose infusions resulted in similar degrees of acyl ghrelin suppression (P>0.05). Direct exposure of the proximal jejunum to glucose increases acyl ghrelin suppression independent of circulating glucose levels. The enhanced suppression of acyl ghrelin after RYGB may be due to a nutrient-initiated signal in the jejunum that regulates ghrelin secretion.

    June 07, 2016   doi: 10.1152/ajpendo.00082.2016   open full text
  • A single session of neuromuscular electrical stimulation does not augment postprandial muscle protein accretion.
    Dirks, M. L., Wall, B. T., Kramer, I. F., Zorenc, A. H., Goessens, J. P. B., Gijsen, A. P., van Loon, L. J. C.
    AJP Endocrinology and Metabolism. June 07, 2016

    Background: The loss of muscle mass and strength that occurs with aging, termed sarcopenia, has been (at least partly) attributed to an impaired muscle protein synthetic response to food intake. We previously showed that neuromuscular electrical stimulation (NMES) can stimulate fasting muscle protein synthesis rates and prevent muscle atrophy during disuse. We hypothesized that NMES prior to protein ingestion would increase postprandial muscle protein accretion. Methods: Eighteen healthy, elderly (69±1 y) males participated in this study. After performing a 70 min unilateral NMES protocol, subjects ingested 20 g intrinsically L-[1-13C]-phenylalanine-labeled casein. Plasma samples and muscle biopsies were collected to assess postprandial mixed muscle and myofibrillar protein accretion, as well as associated myocellular signaling, during a 4 hour post-prandial period in both the control (CON) and stimulated (NMES) leg. Results: Protein ingestion resulted in rapid increases in both plasma phenylalanine concentrations and L-[1-13C]-phenylalanine enrichments, which remained elevated during the entire 4 h postprandial period (P<0.05). Mixed muscle protein bound L-[1-13C]-phenylalanine enrichments significantly increased over time following protein ingestion, with no differences between the CON (0.0164±0.0019 MPE) and NMES (0.0164±0.0019 MPE) leg (P>0.05). In agreement, no differences were observed in the post-prandial rise in myofibrillar protein bound L-[1-13C]-phenylalanine enrichments between the CON and NMES legs (0.0115±0.0014 vs 0.0133±0.0013 MPE, respectively; P>0.05). Significant increases in mTOR and P70S6K phosphorylation status were observed in the NMES stimulated leg only (P<0.05). Conclusion: A single session of NMES prior to food intake does not augment post-prandial muscle protein accretion in healthy, older men.

    June 07, 2016   doi: 10.1152/ajpendo.00085.2016   open full text
  • Maternal Obesity is Associated with Ovarian Inflammation and Up-regulation of Early Growth Response Factor (Egr)-1.
    Ruebel, M., Shankar, K., Gaddy, D., Lindsey, F., Badger, T., Andres, A.
    AJP Endocrinology and Metabolism. June 07, 2016

    Obesity impairs reproductive functions through multiple mechanisms, possibly through disruption of ovarian function. We hypothesized that increased adiposity will lead to a pro-inflammatory gene signature and up-regulation of Egr-1 protein in ovaries from obese (OB, n=7) compared to lean (LN, n=10) female Sprague Dawley rats during the peri-implantation period at 4.5 days post-coitus (dpc). Obesity was induced by overfeeding (40% excess calories for 28 d) via total enteral nutrition prior to mating. OB dams had higher body weight (p<0.001), greater fat mass (p<0.001), reduced lean mass (p<0.05), and developed metabolic dysfunction with elevated serum lipids, insulin, leptin, and CCL2 (p<0.05) compared to LN dams. Microarray analyses identified 284 differentially-expressed genes between ovaries from LN vs. OB dams (±1.3 fold, p<0.05). RT-qPCR confirmed a decrease in expression of glucose transporters GLUT4 and GLUT9 and elevation of pro-inflammatory genes including CCL2, CXCL10, CXCL11, CCR2, CXCR1, and TNF-α in ovaries from OB compared to LN (p<0.05). Protein levels of PI3K and phosphorylated AKT were significantly decreased (p<0.05) while nuclear levels of Egr-1 (p<0.05) were increased in OB compared to LN ovaries. Moreover, Egr-1 was localized to granulosa cells, with highest expression in cumulus cells of pre-ovulatory follicles. mRNA expression of VCAN, AURKB and PLAT (p<0.05) correlated with %visceral fat weight (r=0.51, -0.77, and -0.57, p<0.05, respectively), suggesting alterations in ovarian function with obesity. In summary, maternal obesity led to an up-regulation of inflammatory genes and Egr-1 expression in peri-implantation ovarian tissue, and a concurrent down-regulation of GLUTs and AKT and PI3K protein levels.

    June 07, 2016   doi: 10.1152/ajpendo.00524.2015   open full text
  • Glucose uptake saturation explains glucose kinetics profiles measured by different tests.
    Bizzotto, R., Natali, A., Gastaldelli, A., Muscelli, E., Kr&#x0161;&#x0161;ak, M., Brehm, A., Roden, M., Ferannini, E., Mari, A.
    AJP Endocrinology and Metabolism. May 31, 2016

    It is known that for a given insulin level glucose clearance depends on glucose concentration. However, a quantitative representation of the concomitant effects of hyperinsulinemia and hyperglycemia on glucose clearance, necessary to describe heterogeneous tests such as euglycemic and hyperglycemic clamps and oral tests, is lacking. Data from five studies (123 subjects) using a glucose tracer and including all the above tests in normal and diabetic subjects were collected. A mathematical model was developed in which glucose utilization was represented as a Michaelis-Menten function of glucose with constant Km and insulin-controlled Vmax, consistently with the basic notions of glucose transport. Individual values for the model parameters were estimated using a population approach. Tracer data were accurately fitted in all tests. The estimated Km was 3.88 [2.83 - 5.32] mmol/L (median [interquartile range]). Median model-derived glucose clearance at 600 pmol/L insulin was reduced from 246 to 158 ml min-1 m-2 when glucose was raised from 5 to 10 mmol/L. The model reproduced the characteristic lack of increase in glucose clearance when moderate hyperinsulinemia was accompanied by hyperglycemia. In all tests, insulin sensitivity was inversely correlated with BMI, as expected (R2 = 0.234, p = 0.0001). In conclusion, glucose clearance in euglycemic and hyperglycemic clamps and oral tests can be described with a unifying model, consistent with the notions of glucose transport and able to reproduce the suppression of glucose clearance due to hyperglycemia observed in previous studies. The model may be important for the design of reliable glucose homeostasis simulators.

    May 31, 2016   doi: 10.1152/ajpendo.00045.2016   open full text
  • Hepcidin and 1,25(OH)2D3 effectively restore Ca2+ transport in {beta}-thalassemic mice: reciprocal phenomenon of Fe2+ and Ca2+ absorption.
    Kraidith, K., Svasti, S., Teerapornpuntakit, J., Vadolas, J., Chaimana, R., Lapmanee, S., Suntornsaratoon, P., Krishnamra, N., Fucharoen, S., Charoenphandhu, N.
    AJP Endocrinology and Metabolism. May 31, 2016

    Previously, β-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of β-globin gene, has been reported to induce osteopenia and impaired whole-body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how β-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or hepcidin, the latter of which was liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous β-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermate, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice, which could be restored by 7-day 1,25(OH)2D3 treatment. Such the 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, PMCA1b, as well as vesicular transport inhibitor. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors, MG132 and bortezomib. In conclusion, 1,25(OH)2D3 impairment of calcium absorption. Our study has, therefore, shed light on the development of treatment strategy to rescue calcium dysregulation in β-thalassemia.

    May 31, 2016   doi: 10.1152/ajpendo.00067.2016   open full text
  • The effects of paternal high-fat diet exposure on offspring metabolism with epigenetic changes in the mouse Adiponectin and Leptin gene promoter.
    Masuyama, H., Mitsui, T., Eguchi, T., Tamada, S., Hiramatsu, Y.
    AJP Endocrinology and Metabolism. May 31, 2016

    Recent studies have demonstrated that epigenetic changes resulting from malnutrition might play important roles in transgenerational links with metabolic diseases. We previously observed that exposure to a high fat diet (HFD) in utero caused a metabolic syndrome-like phenomenon through epigenetic modifications of the Adiponectin and Leptin genes that persisted for multiple generations. Recent etiological studies indicated that paternal BMI had effects on offspring BMI that were independent of but additive to maternal BMI effects. Thus, we examined whether paternal HFD-induced obesity affected the metabolic status of offspring through epigenetic changes in the Adiponectin and Leptin genes. Additionally, we investigated whether a normal diet during subsequent generations abolished the epigenetic changes associated with paternal HFD exposure before conception. We observed the effects of paternal HFD exposure before conception over multiple generations on offspring metabolic traits, including weight and fat gain, glucose intolerance, hypertriglyceridemia, abnormal adipocytokine levels, hypertension, and Adiponectin and Leptin gene expression and epigenetic changes. Normal diet consumption by male offspring during the subsequent generation following paternal HFD exposure diminished, while consumption for two generations completely abolished, the effect of paternal HFD exposure on metabolic traits and adipocytokine promoter epigenetic changes in the offspring. The effects of paternal HFD exposure on offspring were relatively weaker than those following HFD exposure in utero. However, paternal HFD exposure had an additive metabolic effect for two generations, suggesting that both paternal and maternal nutrition might affect offspring metabolism through epigenetic modifications of adipocytokine genes for multiple generations.

    May 31, 2016   doi: 10.1152/ajpendo.00095.2016   open full text
  • Intake of branched-chain or essential amino acids attenuates the elevation in muscle levels of PGC-1{alpha}4 mRNA caused by resistance exercise.
    Samuelsson, H., Moberg, M., Apro, W., Ekblom, B., Blomstrand, E.
    AJP Endocrinology and Metabolism. May 31, 2016

    The transcriptional co-activator PGC-1α is recognized as the master regulator of mitochondrial biogenesis. However, recently a novel isoform, PGC-1α4 that specifically regulates muscle hypertrophy was discovered. Since stimulation of mTORC1 activity is tightly coupled to hypertrophy, we hypothesized that activation of this pathway would upregulate PGC-1α4. Eight male subjects performed heavy resistance exercise (10 x 8-12 repetitions at ~75% of 1RM in leg press) on four different occasions, ingesting in random order a solution containing essential amino acids (EAA), branched-chain amino acids (BCAA), leucine or flavored water (placebo) during and after the exercise. Biopsies were taken from the vastus lateralis muscle before and immediately after exercise, as well as following 90 and 180 min of recovery. Signaling through mTORC1, as reflected in S6K1 phosphorylation, was stimulated to a greater extent by the EAA and BCAA than the leucine or placebo supplements. Unexpectedly, intake of EAA or BCAA attenuated the stimulatory effect of exercise on PGC-1α4 expression by ~50% (from a 10-fold to 5-fold increase with BCAA and EAA, P<0.05) 3 h after exercise, whereas intake of leucine alone did not reduce this response. The 60% increase (P<0.05) in the level of PGC-1α1 mRNA 90 min after exercise was uninfluenced by amino acid intake. Muscle glycogen levels were reduced and AMPKα2 activity and phosphorylation of p38 MAPK enhanced to the same extent with all four supplements. In conclusion, induction of PGC-1α4 does not appear to regulate the nutritional (BCAA or EAA) mediated activation of mTORC1 in human muscle.

    May 31, 2016   doi: 10.1152/ajpendo.00154.2016   open full text
  • Differential regulation of lipid and protein metabolism in obese versus lean subjects before and after a 72 h fast.
    Bak, A. M., Moller, A. B., Vendelbo, M. H., Nielsen, T. S., Viggers, R., Rungby, J., Pedersen, S., Jorgensen, J. O. L., Jessen, N., Moller, N.
    AJP Endocrinology and Metabolism. May 31, 2016

    Increased availability of lipids may conserve muscle protein during catabolic stress. Our study was designed to define (i) intracellular mechanisms leading to increased lipolysis and (ii) whether this scenario is associated with decreased amino acid and urea fluxes and decreased muscle amino acid release in obese subjects under basal and fasting conditions. We therefore studied 9 lean and 9 obese subjects twice, after 12 and 72 h of fasting, using measurements of mRNA and protein expression and phosphorylation of lipolytic and protein metabolic signaling molecules in fat and muscle together with whole body and forearm tracer techniques. Obese subjects displayed increased whole body lipolysis, decreased urea production rates and decreased forearm muscle protein breakdown per 100 ml forearm, differences which persisted after 72-h of fasting. Lipolysis per fat mass unit was reduced in obese subjects and correspondingly, adipose tissue HSL phosphorylations and mRNA and protein levels of the ATGL coactivator CGI58 were decreased. Fasting increased HSL phosphorylations and decreased protein levels of the ATGL inhibitor G0S2. Muscle protein expressions of mTOR and 4EBP1 were decreased in obese subjects and MuRf1 mRNA increased with fasting in lean but not obese subjects. mTOR phosphorylation and signaling decreased with fasting in both groups, while ULK1 protein and mRNA levels increased. In summary obese subjects exhibit increased lipolysis, due to a large fat mass with blunted pro-lipolytic signaling, together with decreased urea and amino acid fluxes both in the basal and 72h-fasted state; this is compatible with preservation of muscle and whole body protein.

    May 31, 2016   doi: 10.1152/ajpendo.00464.2015   open full text
  • Mathematical Modeling of the Circadian Dynamics of the Neuroendocrine-Immune Network in Experimentally Induced Arthritis.
    Rao, R. T., DuBois, D. C., Almon, R. R., Jusko, W. J., Androulakis, I. P.
    AJP Endocrinology and Metabolism. May 24, 2016

    The circadian dynamics of important neuroendocrine-immune mediators have been implicated in progression of rheumatoid arthritis pathophysiology both clinically as well as in animal models. We present a mathematical model that describes the circadian interactions between mediators of the HPA axis and the proinflammatory cytokines. Model predictions demonstrate that chronically elevated cytokine expression results in the development of adrenal insufficiency and circadian variability in paw edema progression. Notably, our model also predicts that an increase in mean secretion of CST after the induction of the disease is accompanied by a decrease in the amplitude of the CST oscillation. Furthermore, alterations in the phase of circadian oscillation of both cytokines and HPA axis mediators are observed. Therefore, by incorporating the circadian interactions between the neuroendocrine-immune mediators our model is able to simulate important features of rheumatoid arthritis pathophysiology.

    May 24, 2016   doi: 10.1152/ajpendo.00006.2016   open full text
  • {beta}-cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion.
    Reynolds, M. S., Hancock, C. R., Ray, J. D., Kener, K. B., Draney, C., Garland, K., Hardman, J., Bikman, B. T., Tessem, J. S.
    AJP Endocrinology and Metabolism. May 24, 2016

    Iβ-cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle and adipose. We have previously demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic β-cells. In this study we examined whether Nr4a expression impacts pancreatic β-cell mitochondrial function. Here we show that β-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in β-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP, and ultimately inhibits glucose stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for β-cell mitochondrial function and insulin secretion.

    May 24, 2016   doi: 10.1152/ajpendo.00022.2016   open full text
  • Liver-derived IGF-I regulates cortical bone mass but is dispensable for the osteogenic response to mechanical loading in female mice.
    Svensson, J., Windahl, S. H., Saxon, L. K., Sjogren, K., Koskela, A., Tuukkanen, J., Ohlsson, C.
    AJP Endocrinology and Metabolism. May 24, 2016

    Low circulating IGF-I is associated with increased fracture risk. Conditional depletion of IGF-I produced in osteoblasts or osteocytes inhibits the bone anabolic effect of mechanical loading. Here, we determined the role of endocrine IGF-I for the osteogenic response to mechanical loading in young adult and old female mice with adult, liver-specific IGF-I inactivation (LI-IGF-I-/- mice, serum IGF-I reduced by 70%) and control mice. The right tibia was subjected to short periods of axial cyclic compressive loading, 3 times a week for 2 weeks, and measurements were performed using micro-computed tomography (µCT) and mechanical testing by three-point bending. In the non-loaded left tibia, the LI-IGF-I-/- mice had lower cortical bone area and increased cortical porosity, resulting in reduced bone mechanical strength compared to the controls. Mechanical loading induced a similar response in LI-IGF-I-/- and control mice in terms of cortical bone area and trabecular bone volume fraction (BV/TV). In fact, mechanical loading produced a more marked increase in cortical bone mechanical strength, associated with a less marked increase in cortical porosity, in the LI-IGF-I-/- mice compared to the control mice. In conclusion, liver-derived IGF-I regulates cortical bone mass, cortical porosity, and mechanical strength under normal (non-loaded) conditions. However, despite an approximately 70% reduction in circulating IGF-I, the osteogenic response to mechanical loading was not attenuated in the LI-IGF-I-/- mice.

    May 24, 2016   doi: 10.1152/ajpendo.00107.2016   open full text
  • Sodium nitrate co-ingestion with protein does not augment postprandial muscle protein synthesis rates in older, type 2 diabetes patients.
    Kouw, I. W. K., Cermak, N. M., Burd, N. A., Churchward-Venne, T. A., Senden, J. M. G., Gijsen, A. P., van Loon, L. J. C.
    AJP Endocrinology and Metabolism. May 24, 2016

    Introduction: The age-related anabolic resistance to protein ingestion is suggested to be associated with impairments in insulin-mediated capillary recruitment and postprandial muscle tissue perfusion. The present study investigated whether dietary nitrate co-ingestion with protein improves myofibrillar protein synthesis in older, type 2 diabetes patients. Methods: Twenty-four males with type 2 diabetes (age 72±1 y; BMI 26.7±1.4 m·kg-2; HbA1C 7.3±0.4%) received a primed continuous infusion of L-[ring-2H5]-phenylalanine and L-[1-13C]-leucine and ingested 20 g intrinsically L-[1-13C]-phenylalanine and L-[1-13C]-leucine labeled protein with (PRONO3) or without (PRO) sodium nitrate (NaNO3-; 0.15 mmol·kg-1). Blood and muscle samples were collected to assess protein digestion and absorption kinetics and postprandial muscle protein synthesis rates. Results: Upon protein ingestion, exogenous phenylalanine appearance rates increased in both groups (P<0.001) resulting in 55±2% and 53±2% of dietary protein-derived amino acids becoming available in the circulation over the 5h postprandial period in the PRO and PRONO3 group, respectively. Postprandial myofibrillar protein synthesis rates based on L-[ring-2H5]-phenylalanine did not differ between groups (0.025±0.004 vs 0.021±0.007%.h-1 over 0-2h and 0.032±0.004 vs 0.030±0.003%.h-1 over 2-5h in the PRO and PRONO3 group, respectively; ,P=0.7). No differences were observed in the incorporation of dietary protein-derived L-[1-13C]-phenylalanine into de novo myofibrillar protein at 5h (0.016±0.002 vs 0.014±0.002 MPE, in PRO and PRONO3, respectively; P=0.8). Conclusion: Dietary nitrate co-ingestion with protein does not modulate digestion and absorption kinetics, and does not further increase postprandial muscle protein synthesis rates or the incorporation of dietary protein-derived amino acids into de novo myofibrillar protein in older, type 2 diabetes patients.

    May 24, 2016   doi: 10.1152/ajpendo.00122.2016   open full text
  • Insulin Decreases Atherosclerotic Plaque Burden and Increases Plaque Stability via Nitric Oxide Synthase in Apolipoprotein E-null Mice.
    Mori, Y., Chiang, S., Bendeck, M. P., Giacca, A.
    AJP Endocrinology and Metabolism. May 24, 2016

    It has been argued whether insulin accelerates or prevents atherosclerosis. Although results from in vitro studies have been conflicting, recent in vivo mice studies demonstrated anti-atherogenic effects of insulin. Insulin is a known activator of endothelial nitric oxide synthase (NOS), leading to increased production of NO, which has potent anti-atherogenic effects. We aimed to examine the role of NOS in the protective effects of insulin against atherosclerosis. Male apolipoprotein E-null mice (8wk old) fed high cholesterol diet (1.25% cholesterol) were assigned to the following 12wk treatments: control, insulin (0.05U/day via subcutaneous pellet), N-Nitro-L-arginine methyl ester hydrochloride (L-NAME, via drinking water at 100mg/l), and insulin plus L-NAME. Insulin reduced atherosclerotic plaque burden in the descending aorta by 42% compared to control (plaque area / aorta lumen area: control, 16.5±1.9%; insulin, 9.6±1.3%, p<0.05). Although insulin did not decrease plaque burden in the aortic sinus, macrophage accumulation in the plaque was decreased by insulin. Furthermore insulin increased smooth muscle actin and collagen content, and decreased plaque necrosis, consistent with increased plaque stability. In addition, insulin treatment increased plasma NO levels, decreased iNOS staining and tended to increase phosphorylated vasodilator-stimulated phosphoprotein staining in the plaques of the aortic sinus. All these effects of insulin were abolished by co-administration of L-NAME, while L-NAME alone showed no effect. Insulin also tended to increase phoshorylated endothelial NOS and total neuronal NOS staining, effects not modified by L-NAME. In conclusion, we demonstrated that insulin treatment decreases atherosclerotic plaque burden and increases plaque stability through NOS dependent mechanisms.

    May 24, 2016   doi: 10.1152/ajpendo.00320.2015   open full text
  • Age-related impairments in skeletal muscle PDH phosphorylation and plasma lactate are indicative of metabolic inflexibility and the effects of exercise training.
    Consitt, L. A., Saxena, G., Saneda, A., Houmard, J. A.
    AJP Endocrinology and Metabolism. May 24, 2016

    The purpose of this study was to determine if plasma lactate and skeletal muscle glucose regulatory pathways, specifically PDH dephosphorylation, are impaired during hyperinsulinemic conditions in middle- to older-aged individuals, and determine if exercise training could improve key variables responsible for skeletal muscle PDH regulation. Eighteen young (19-29 years, n=9/9, male/female) and twenty middle- to older-aged (57-82 years, n=10/10, male/female) underwent a 2-hr euglycemic-hyperinsulinemic clamp. Plasma samples were obtained at baseline, 30 min, 50 min, 90 min, and 120 min for analysis of lactate and skeletal muscle biopsies were performed at 60 min for analysis of protein associated with glucose metabolism. In response to insulin, plasma lactate was elevated in aged individuals when normalized to insulin action. Insulin-stimulated phosphorylation of skeletal muscle PDH on serine sites 232, 293, and 300 decreased in young individuals only. Changes in insulin-stimulated PDH phosphorylation was positively related to changes in plasma lactate. No age-related differences were observed in skeletal muscle phosphorylation of LDH, GSK3α, or GSK3β in response to insulin, or PDP1, PDP2, PDK2, PDK4 or MPC1 total protein. Twelve weeks of endurance- or strength-oriented exercise training, improved insulin-stimulated PDH dephosphorylation which was related to a reduced lactate response. These findings suggest that impairments in insulin-induced PDH regulation in a sedentary aging population contribute to impaired glucose metabolism and that exercise training is an effective intervention for treating metabolic inflexibility.

    May 24, 2016   doi: 10.1152/ajpendo.00452.2015   open full text
  • MiR-206 is Expressed in Pancreatic Islets and Regulates Glucokinase Activity.
    Vinod, M., Patankar, J. V., Sachdev, V., Frank, S., Graier, W. F., Kratky, D., Kostner, G. M.
    AJP Endocrinology and Metabolism. May 24, 2016

    Glucose homeostasis is a complex indispensable process and its dysregulation causes hyperglycaemia and type 2 diabetes mellitus. Glucokinase (GK) takes a central role in these pathways and thus is rate-limiting for glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Several reports have described the transcriptional regulation of Gck mRNA, while its post-transcriptional mechanisms of regulation, especially those involving microRNAs (miR), are poorly understood. In this study, we investigated the role of miR-206 as a post-transcriptional regulator of Gck. In addition, we examined the effects of miR-206 on glucose tolerance, GSIS, and gene expression in control and germ line miR-206 knock-out (KO) mice fed either with chow or high-fat diet (HFD). MiR-206 was found in Gck-expressing tissues and was differentially altered in response to HFD-feeding. Pancreatic islets showed the most profound induction in the expression of miR-206 in response to HFD. Chow- and HFD-fed miR-206 KO mice have improved glucose tolerance and GSIS but unaltered insulin sensitivity. In silico analysis of Gck mRNA revealed a conserved 8-mer miR-206 binding site. Hence, the predicted regulation of Gck by miR-206 was confirmed in reporter and GK activity assays. Concomitant with increased GK activity, miR-206KO mice had elevated liver glycogen content and plasma lactate concentrations. Our findings revealed a novel mechanism of post-transcriptional regulation of Gck by miR-206 and underline the crucial role of pancreatic islet miR-206 in the regulation of whole-body glucose homeostasis in a murine model that mimics the metabolic syndrome.

    May 24, 2016   doi: 10.1152/ajpendo.00510.2015   open full text
  • Insights Into The Role Of Neuronal Glucokinase.
    De Backer, I., Hussain, S. S., Bloom, S. R., Gardiner, J. V.
    AJP Endocrinology and Metabolism. May 17, 2016

    Glucokinase is a key component of the neuronal glucose-sensing mechanism and is expressed in brain regions that control a range of homeostatic processes. In this review, we detail recently identified roles for neuronal glucokinase in glucose homeostasis and counter-regulatory responses to hypoglycaemia and in regulating appetite. We describe clinical implications from these advances in our knowledge especially for developing novel treatments for diabetes and obesity. Further research required to extend our knowledge and help our efforts to tackle the diabetes and obesity epidemics are suggested.

    May 17, 2016   doi: 10.1152/ajpendo.00034.2016   open full text
  • Emerging Role for Regulated in Development and DNA Damage 1 (REDD1) in the Regulation of Skeletal Muscle Metabolism.
    Gordon, B. S., Steiner, J. L., Williamson, D. L., Lang, C. H., Kimball, S. R.
    AJP Endocrinology and Metabolism. May 17, 2016

    Since its discovery, the protein Regulated in Development and DNA Damage 1 (REDD1) has been implicated in the cellular response to various stressors. Most notably, its role as a repressor of signaling through the central metabolic regulator, the mechanistic target of rapamycin in complex 1 (mTORC1) has gained considerable attention. Not surprisingly, changes in REDD1 mRNA and protein have been observed in skeletal muscle under various physiological conditions (e.g., nutrient consumption and resistance exercise) and pathological conditions (e.g., sepsis, alcoholism, diabetes, obesity) suggesting a role for REDD1 in regulating mTORC1-dependent skeletal muscle protein metabolism. Our understanding of the causative role of REDD1 in skeletal muscle metabolism is increasing mostly due to the availability of genetically modified mice in which the REDD1 gene is disrupted. Results from such studies provide support for an important role for REDD1 in the regulation of mTORC1 as well as reveal unexplored functions of this protein in relation to other aspects of skeletal muscle metabolism. The goal of this work is to provide a comprehensive review of the role of REDD1 (and its paralog REDD2) in skeletal muscle during both physiological and pathological conditions.

    May 17, 2016   doi: 10.1152/ajpendo.00059.2016   open full text
  • Perilipin 5 is dispensable for normal substrate metabolism and in the adaptation of skeletal muscle to exercise training.
    Mohktar, R. A. M., Montgomery, M. K., Murphy, R. M., Watt, M. J.
    AJP Endocrinology and Metabolism. May 17, 2016

    Cytoplasmic lipid droplets provide a reservoir for triglyceride storage and are a central hub for fatty acid trafficking in cells. The protein perilipin (PLIN) 5 is highly expressed in oxidative tissues such as skeletal muscle and regulates lipid metabolism by coordinating the trafficking and the reversible interactions of effector proteins at the lipid droplet. PLIN5 may also regulate mitochondrial function, although this remains unsubstantiated. Hence, the aims of this study were to examine the role of PLIN5 in the regulation of skeletal muscle substrate metabolism during acute exercise and to determine whether PLIN5 is required for the metabolic adaptations and enhancement in exercise tolerance following endurance exercise training. Using muscle-specific Plin5 knockout mice (Plin5MKO), we show that PLIN5 is dispensable for normal substrate metabolism during exercise as reflected by levels of blood metabolites and rates of glycogen and triglyceride depletion that were indistinguishable from control (lox/lox) mice. Plin5MKO mice exhibited a functional impairment in their response to endurance exercise training as reflected by reduced maximal running capacity (20%) and reduced time to fatigue during prolonged submaximal exercise (15%). The reduction in exercise performance was not accompanied by alterations in carbohydrate and fatty acid metabolism during submaximal exercise. Similarly, mitochondrial capacity (mtDNA, respiratory complex proteins, citrate synthase activity) and mitochondrial function (oxygen consumption rate in muscle fiber bundles) was not different between lox/lox and Plin5MKO mice. Thus, PLIN5 is dispensable for normal substrate metabolism during exercise and is not required to promote mitochondrial biogenesis or enhance the cellular adaptations to endurance-exercise training.

    May 17, 2016   doi: 10.1152/ajpendo.00084.2016   open full text
  • No insulating effect of obesity.
    Fischer, A. W., Csikasz, R., von Essen, G., Cannon, B., Nedergaard, J.
    AJP Endocrinology and Metabolism. May 17, 2016

    The development of obesity may be aggravated if obesity itself insulates against heat loss and thus diminishes the amount of food burnt for body temperature control. This would be particularly important under normal animal house conditions where mice experience a chronic cold stress (at 20 °C). We used Scholander plots (energy expenditure plotted versus ambient temperature) to examine the insulation (thermal conductance) of mice, defined as the slope of the Scholander curve at subthermoneutral temperatures. We verified the method by demonstrating that shaved mice possessed only half the insulation of nonshaved mice. We examined a series of obesity models (mice fed high-fat diets and kept at different temperatures, classical diet-induced obesity mice, ob/ob mice and obesity-prone (C57BL/6) versus obesity-resistant (129S) mice). We found that neither acclimation temperature, nor any kind or degree of obesity affected the thermal insulation of the mice, when analyzed at the whole mouse level or as energy expenditure per lean weight. Calculation per body weight erroneously implied increased insulation in obese mice. We conclude that in contrast to what would be expected, obesity of any kind does not increase thermal insulation in mice, and it does therefore not in itself aggravate the development of obesity. It may be discussed to what degree excess adipose tissue has an insulation effect in humans and especially whether significant metabolic effects are associated with insulation in humans.

    May 17, 2016   doi: 10.1152/ajpendo.00093.2016   open full text
  • Neuroinflammatory and autonomic mechanisms in diabetes and hypertension.
    Han, C., Rice, M. W., Cai, D.
    AJP Endocrinology and Metabolism. May 10, 2016

    Interdisciplinary studies in the research fields of endocrinology and immunology show that obesity-associated overnutrition leads to neuroinflammatory molecular changes, in particular in the hypothalamus, chronically causing various disorders known as elements of metabolic syndrome. In this process, neural or hypothalamic inflammation impairs the neuroendocrine and autonomic regulation of the brain over blood pressure and glucose homeostasis as well as insulin secretion, and elevated sympathetic activation was generally appreciated responsible. This review describes the involved physiology and mechanisms, with a focus on glucose and blood pressure balance, and suggests that neuroinflammation employs the autonomic nervous system to mediate the development of diabetes and hypertension.

    May 10, 2016   doi: 10.1152/ajpendo.00012.2016   open full text
  • Reduced intestinal lipid absorption and body weight-independent improvements in insulin sensitivity in high-fat diet-fed Park2 knockout mice.
    Costa, D. K., Huckestein, B. R., Edmunds, L. R., Petersen, M. C., Nasiri, A., Butrico, G. M., Abulizi, A., Harmon, D. B., Lu, C., Mantell, B. S., Hartman, D. J., Camporez, J.-P. G., O'Doherty, R. M., Cline, G. W., Shulman, G. I., Jurczak, M. J.
    AJP Endocrinology and Metabolism. May 10, 2016

    Mitochondrial dysfunction is associated with many human diseases and results from mismatch of damage and repair over the life of the organelle. Park2 is an ubiquitin E3 ligase that regulates mitophagy, a repair mechanism that selectively degrades damaged mitochondria. Deletion of Park2 in multiple in vivo models results in susceptibility to stress-induced mitochondrial and cellular dysfunction. Surprisingly, Park2 knockout (KO) mice are protected from nutritional stress and do not develop obesity, hepatic steatosis or insulin resistance when fed high-fat diet (HFD). However, these phenomena are casually related and the physiological basis for this phenotype is unknown. We therefore undertook a series of acute HFD studies to more completely understand the physiology of Park2 KO during nutritional stress. We find that intestinal lipid absorption is impaired in Park2 KO mice as evidenced by increased fecal lipids and reduced plasma triglycerides after intragastric fat challenge. Park2 KO mice developed hepatic steatosis in response to intravenous lipid infusion, as well as during incubation of primary hepatocytes with fatty acids, suggesting that hepatic protection from nutritional stress was secondary to changes in energy balance due to altered intestinal triglyceride absorption. Park2 KO mice showed reduced adiposity after one-week HFD, as well as improved hepatic and peripheral insulin sensitivity. These studies suggest that changes in intestinal lipid absorption may play a primary role in protection from nutritional stress in Park2 KO mice by preventing HFD-induced weight gain and highlight the need for tissue-specific models to address the role of Park2 during metabolic stress.

    May 10, 2016   doi: 10.1152/ajpendo.00042.2016   open full text
  • Lack of Mitochondria-generated acetyl-CoA by pyruvate dehydrogenase complex downregulates gene expression in hepatic de novo lipogenic pathway.
    Mahmood, S., Birkaya, B., Rideout, T. C., Patel, M. S.
    AJP Endocrinology and Metabolism. May 10, 2016

    During the absorptive state the liver stores excess glucose as glycogen and synthesizes fatty acids for triglyceride synthesis for export as very low density lipoproteins. For de novo synthesis of fatty acids from glucose, the mitochondrial pyruvate dehydrogenase complex (PDC) is the gatekeeper for the generation of acetyl-CoA from glucose-derived pyruvate. Here we tested the hypothesis that limiting the supply of PDC-generated acetyl-CoA from glucose would have an impact on expression of key genes in the lipogenic pathway. In the present study, although the postnatal growth of liver-specific PDC-deficient (L-PDCKO) male mice was largely unaltered, they developed hyperinsulinemia with lower blood glucose levels in the fed state. Serum and liver lipid triglyceride and cholesterol levels remained unaltered in L-PDCKO mice. Expression of several key genes (ACL, ACC1) in the lipogenic pathway and their upstream regulators (LXR, SREBP1, ChREBP) as well as several genes in glucose metabolism (Pklr, G6pd2, Pck1) and fatty acid oxidation (FAT, Cpt1a) was downregulated in livers from L-PDCKO mice. Although, the total hepatic acetyl-CoA content remained unaltered in L-PDCKO mice, modified acetylation profiles of proteins in the nuclear compartment suggested an important role of PDC-generated acetyl-CoA in gene expression in de novo fatty acid synthesis in the liver. This finding has important implications for regulation of hepatic lipid synthesis in pathological states.

    May 10, 2016   doi: 10.1152/ajpendo.00064.2016   open full text
  • Hypothalamic Roles of mTOR Complex I: Integration of nutrient and hormone signals to regulate energy homeostasis.
    Hu, F., Xu, Y., Liu, F.
    AJP Endocrinology and Metabolism. May 10, 2016

    Mammalian or mechanistic target of rapamycin (mTOR) senses nutrient, energy, and hormone signals to regulate metabolism and energy homeostasis. mTOR activity in the hypothalamus, which is associated with changes in energy status, plays a critical role in the regulation of food intake and body weight. mTOR integrates signals from a variety of "energy balancing" hormones such as leptin, insulin, and ghrelin, although its action varies in response to these distinct hormonal stimuli as well as across different neuronal populations. In this review, we summarize and highlight recent findings regarding the functional roles of mTOR complex 1 (mTORC1) in the hypothalamus specifically in its regulation of body weight, energy expenditure and glucose/lipid homeostasis. Understanding the role and underlying mechanisms behind mTOR-related signaling in the brain will undoubtedly pave new avenues for future therapeutics and interventions that can combat obesity, insulin resistance, and diabetes.

    May 10, 2016   doi: 10.1152/ajpendo.00121.2016   open full text
  • AP2-NR4A3 transgenic mice display reduced serum epinephrine due to increased catecholamine catabolism in adipose tissue.
    Walton, R. G., Zhu, X., Tian, L., Heywood, E. B., Liu, J., Hill, H. S., Lamiquiz, J. L., Bruemmer, D., Yang, Q., Fu, Y., Garvey, W. T.
    AJP Endocrinology and Metabolism. May 10, 2016

    The NR4A orphan nuclear receptors function as early response genes to numerous stimuli. Our laboratory has previously demonstrated that over-expression of NR4A3 (NOR-1, MINOR) in 3T3-L1 adipocytes enhances insulin-stimulated glucose uptake. To assess the in vivo effect of NR4A3 on adipocytes, we generated transgenic mice with NR4A3 over-expression driven by the adipocyte AP2 promoter (AP2-NR4A3 mice). We hypothesized that AP2-NR4A3 mice would display enhanced glucose tolerance and insulin sensitivity. However, AP2-NR4A3 mice exhibit metabolic impairment, including increased fasting glucose and insulin, impaired glucose tolerance, insulin resistance, decreased serum free fatty acids, and increased LDL-cholesterol. Furthermore, AP2-NR4A3 mice display a significant reduction in serum epinephrine due to increased expression of catecholamine catabolizing enzymes in adipose tissue, including monoamine oxidase-A. Furthermore, enhanced expression of monoamine oxidase-A is due to direct transcriptional activation by NR4A3. Finally, AP2-NR4A3 mice display cardiac and behavioral alterations consistent with chronically low circulating epinephrine levels. In conclusion, overexpression of NR4A3 in adipocytes produces a complex phenotype characterized by impaired glucose metabolism and low serum catecholamines, due to enhanced degradation by adipose tissue.

    May 10, 2016   doi: 10.1152/ajpendo.00330.2015   open full text
  • Characterizing Active and Inactive Brown Adipose Tissue in Adult Humans Using PET-CT and MR Imaging.
    Gifford, A., Towse, T. F., Walker, R. C., Avison, M. J., Welch, E. B.
    AJP Endocrinology and Metabolism. May 10, 2016

    Activated brown adipose tissue (BAT) plays an important role in thermogenesis and whole-body metabolism in mammals. Positron emission tomography (PET)-computed tomography (CT) imaging has identified depots of BAT in adult humans, igniting scientific interest. The purpose of this present study is to characterize both active and inactive supraclavicular BAT in adults, and compare the values to those of subcutaneous white adipose tissue (WAT). We obtained 18F-fluorodeoxyglucose (18F-FDG) PET-CT and magnetic resonance imaging (MRI) scans of 25 healthy adults. Unlike 18F-FDG PET, which can only detect active BAT, MRI is capable of detecting both active and inactive BAT. The MRI derived fat-signal fraction (FSF) of active BAT was significantly lower than inactive BAT (mean ± SD): 60.2 ± 7.6% vs. 62.4 ± 6.8%, respectively. This change in tissue morphology was also reflected as a significant increase in Hounsfield Units (HU): -69.4 ± 11.5 HU vs. -74.5 ± 9.7 HU, respectively. Additionally, the CT HU, MRI FSF and MRI R2* values are significantly different between BAT and WAT, regardless of the activation status of BAT. To the best of our knowledge this is the first study to quantify PET-CT and MRI measures of BAT in both active and inactive states in the same adult subjects. Our findings support the use of these metrics to characterize and distinguish between BAT and WAT, and lay the foundation for future MRI analysis with the hope that someday MRI-based delineation of BAT can stand on its own.

    May 10, 2016   doi: 10.1152/ajpendo.00482.2015   open full text
  • Na,K-ATPase regulation in skeletal muscle.
    Pirkmajer, S., Chibalin, A. V.
    AJP Endocrinology and Metabolism. May 10, 2016

    Skeletal muscle contains one of the largest and the most dynamic pools of Na,K-ATPase (NKA) in the body. Under resting conditions NKA in skeletal muscle operates at only a fraction of maximal pumping capacity, but it can be markedly activated when demands for ion transport increase, such as during exercise or following food intake. Given the size, capacity, and dynamic range of the NKA pool in skeletal muscle, its tight regulation is essential to maintain whole-body homeostasis as well as muscle function. To reconcile functional needs of systemic homeostasis with those of skeletal muscle, NKA is regulated in a coordinated manner by extrinsic stimuli, such as hormones and nerve-derived factors, as well as by local stimuli arising in skeletal muscle fibers, such as contractions and muscle energy status. These stimuli regulate NKA acutely by controlling its enzymatic activity and/or its distribution between plasma membrane and the intracellular storage compartment. They also regulate NKA chronically by controlling NKA gene expression, thus determining total NKA content in skeletal muscle and its maximal pumping capacity. This review focuses on molecular mechanisms that underlie regulation of NKA in skeletal muscle by major extrinsic and local stimuli. Special emphasis is given to stimuli and mechanisms linking regulation of NKA and energy metabolism in skeletal muscle, such as insulin and the energy-sensing AMP-activated protein kinase. Finally, the recently uncovered roles for glutathionylation, nitric oxide, and extracellular K+ in regulation of NKA in skeletal muscle are highlighted.

    May 10, 2016   doi: 10.1152/ajpendo.00539.2015   open full text
  • Loss of CTRP5 improves insulin action and hepatic steatosis.
    Lei, X., Rodriguez, S., Petersen, P. S., Seldin, M. M., Bowman, C. E., Wolfgang, M. J., Wong, G. W.
    AJP Endocrinology and Metabolism. May 03, 2016

    The gene that encodes C1q/TNF-related protein 5 (CTRP5), a secreted protein of the C1q family, is mutated in individuals with late-onset retinal degeneration. CTRP5 is widely expressed outside the eye and also circulates in plasma. Its physiological role in peripheral tissues, however, has yet to be elucidated. Here, we show that Ctrp5 expression is modulated by fasting and refeeding, and by different diets, in mice. Adipose expression of CTRP5 was markedly upregulated in obese and diabetic humans, and in genetic and dietary models of obesity in rodents. Further, human CTRP5 expression in the subcutaneous fat depot positively correlated with BMI. A genetic loss-of-function mouse model was used to address the metabolic function of CTRP5 in vivo. On a standard chow diet, CTRP5-deficient mice had reduced fasting insulin but were otherwise comparable to wild-type littermate controls in body weight and adiposity. However, when fed a high-fat diet, CTRP5-deficient animals had attenuated hepatic steatosis and improved insulin action. Loss of CTRP5 also improved the capacity of chow-fed aged mice to respond to subsequent high-fat feeding, as evidenced by decreased insulin resistance. In cultured adipocytes and myotubes, recombinant CTRP5 treatment attenuated insulin-stimulated AKT phosphorylation. Our results provide the first genetic and physiological evidence for CTRP5 as a negative regulator of glucose metabolism and insulin sensitivity. Inhibition of CTRP5 action may result in the alleviation of insulin resistance associated with obesity and diabetes.

    May 03, 2016   doi: 10.1152/ajpendo.00010.2016   open full text
  • Increased ADAMTS1 mediates SPARC-dependent collagen deposition in the aging myocardium.
    Toba, H., de Castro Bras, L. E., Baicu, C. F., Zile, M. R., Lindsey, M. L., Bradshaw, A. D.
    AJP Endocrinology and Metabolism. May 03, 2016

    Secreted protein acidic and rich in cysteine (SPARC) is a collagen-binding matricellular protein highly expressed during fibrosis. Fibrosis is a prominent component of cardiac aging that reduces myocardial elasticity. We previously reported that SPARC deletion attenuated myocardial stiffness and collagen deposition in aged mice. To investigate the mechanisms by which SPARC promotes age-related cardiac fibrosis, we evaluated 6 groups of mice (n=5-6/group): young (3-5 month old), middle-aged (10-12 month old) and old (18-29 month old) C57BL/6 wild type (WT) and SPARC-null (Null) mice. Collagen content, determined by picrosirius red staining, increased in an age-dependent manner in WT but not in Null mice. A disintegrin and metalloproteinase with thrombospondin type 1 motif, 1 (ADAMTS1) increased in middle-aged and old WT compared to young, whereas in Null mice, only old animals showed increased ADAMTS1 expression. Versican, a substrate of ADAMTS1, decreased with age only in WT. To assess the mechanisms of SPARC-induced collagen deposition, we stimulated cardiac fibroblasts with SPARC. SPARC treatment increased secretion of collagen I and ADAMTS1 (both the 110kDa latent and 87 kDa active forms) into the conditioned media, as well as increased the cellular expression of transforming growth factor β1-induced protein (Tgfbi) and phosphorylated Smad2. An ADAMTS1 blocking antibody suppressed the SPARC-induced collagen I secretion, indicating that SPARC promoted collagen production directly through ADAMTS1 interaction. In conclusion, ADAMTS1 is an important mediator of SPARC-regulated cardiac aging.

    May 03, 2016   doi: 10.1152/ajpendo.00040.2016   open full text
  • Pre-hepatic secretion and disposal of insulin in obese adolescents estimated from 3-hour, 8-sample oral glucose tolerance tests.
    Vogt, J. A., Domzig, C., Wabitsch, M., Denzer, C.
    AJP Endocrinology and Metabolism. May 03, 2016

    Background Early-stage insulin resistance is compensated for by increased insulin secretion. A reliable and easy-to-use mathematical assessment of insulin secretion and disposal could be a valuable tool to identify patients at risk for the development of type 2 diabetes. Because the pathophysiology of insulin resistance is incompletely understood, assessing insulin metabolism with minimal assumptions regarding the metabolic regulation is a major challenge. Materials and Methods To assess insulin secretion and indexes for insulin disposal, our marginalized and regularized absorption approach (MRA) was applied to a sparse sampling oral glucose tolerance test (OGTT) protocol using insulin and C-peptide concentrations. Identifiability and potential bias of metabolic parameters were estimated from published data with dense sampling. This approach was applied to OGTT data from 135 obese adolescents to demonstrate its clinical applicability. Results Individual prehepatic basal and dynamic insulin secretion and clearance values were determined with a precision and accuracy greater than 10% of the nominal value. The inter-subject variability in these parameters was approximately 4 times higher than the intra-subject variability, and there was a close negative correlation between prehepatic secretion and plasma clearance. Conclusion MRA-based analysis provides reliable estimates of insulin secretion and clearance, thereby enabling detailed glucose homeostasis characterization based on restricted datasets that are obtainable during routine patient care.

    May 03, 2016   doi: 10.1152/ajpendo.00455.2014   open full text
  • Glucocorticoid Antagonism Limits Adiposity Rebound and Glucose Intolerance in Young Male Rats Following the Cessation of Daily Exercise and Caloric Restriction.
    Teich, T., Dunford, E. C., Porras, D. P., Pivovarov, J. A., Beaudry, J. L., Hunt, H., Belanoff, J. K., Riddell, M. C.
    AJP Endocrinology and Metabolism. May 03, 2016

    Severe caloric restriction, in a setting of regular physical exercise, may be a stress that sets the stage for adiposity rebound and insulin resistance when the food restriction and exercise stops. In this study, we examined the effect of mifepristone, a glucocorticoid (GC) receptor antagonist, on limiting adipose tissue mass gain and preserving whole-body insulin sensitivity following the cessation of daily running and caloric restriction (CR). We calorically restricted male Sprague-Dawley rats and provided access to voluntary running wheels for three weeks followed by locking of the wheels and reintroduction to ad libitum feeding with or without mifepristone (80 mg/kg/day) for one week. Cessation of daily running and CR increased HOMA-IR, visceral adipose mass as well as glucose and insulin area under the curve during an oral glucose tolerance test versus pre-wheel lock exercised rats and sedentary rats (all p<0.05). Insulin sensitivity and glucose tolerance were preserved and adipose tissue mass gain was attenuated by daily mifepristone treatment during the post-wheel lock period. These findings suggest that following regular exercise and CR there are GC-induced mechanisms that promote adipose tissue mass gain and impaired metabolic control in healthy organisms and that this phenomenon can be inhibited by the GC receptor antagonist mifepristone.

    May 03, 2016   doi: 10.1152/ajpendo.00490.2015   open full text
  • Vitamin D status and metabolism in an ovine pregnancy model: effect of long-term high-altitude hypoxia.
    Goyal, R., Billings, T. L., Mansour, T., Martin, C., Baylink, D., Longo, L. D., Pearce, W. J., Mata-Greenwood, E.
    AJP Endocrinology and Metabolism. May 03, 2016

    Objective: Vitamin D status increases during healthy mammalian pregnancy, but the molecular determinants remain uncharacterized. The first objective of this study was to determine the effects of pregnancy and the second objective was to examine the role of chronic hypoxia on vitamin D status and metabolism in an ovine model. Approach and Results: We analyzed the plasma levels of cholecalciferol, 25-OH-D, and 1α, 25-(OH)2-D in non-pregnant ewes, near-term pregnant ewes, and their fetuses exposed to normoxia (low altitude) or hypoxia (high-altitude) for 100 days. Hypoxic sheep had increased circulating levels of 25-OH-D and 1α, 25-(OH)2-D compared to normoxic sheep. Hypoxia increases in 25-OH-D were associated with increased expression of renal 25-hydroxylases CYP2R1 and CYP2J. Pregnancy did not further increase the plasma levels of 25-OH-D but it significantly increased those of the active metabolite, 1α, 25-(OH)2-D, in both normoxic and hypoxic ewes. Increased bioactivation of vitamin D correlated with increased expression of the vitamin D activating enzyme, CYP27b1, and decreased expression of the inactivating enzyme CYP24a1 in maternal kidneys and placentas. Hypoxia increased parathyroid hormone levels and further increased renal CYP27b1. Pregnancy and hypoxia decreased the expression of vitamin D receptor (VDR) in maternal kidney and lung with opposite effects on placental VDR. Conclusions: Ovine pregnancy is a model of increased vitamin D status and long-term hypoxia further improves vitamin D status due to pregnancy- and hypoxia-specific regulation of VDR and metabolic enzymes.

    May 03, 2016   doi: 10.1152/ajpendo.00494.2015   open full text
  • Enteral {beta}-Hydroxy-{beta}-Methylbutyrate Supplementation Increases Protein Synthesis in Skeletal Muscle of Neonatal Pigs.
    Kao, M., Columbus, D. A., Suryawan, A., Steinhoff-Wagner, J., Hernandez-Garcia, A., Nguyen, H. V., Fiorotto, M. L., Davis, T. A.
    AJP Endocrinology and Metabolism. May 03, 2016

    Many low birth weight infants are at risk of poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid, leucine, stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite, β-hydroxy-β-methylbutyrate (HMB), on protein synthesis and the regulation of translation initiation and degradation pathways, overnight fasted neonatal pigs were studied immediately (F) or fed 1 of 5 diets for 24 h: low protein (LP), high protein (HP), or LP diet supplemented with 4 (HMB 4), 40 (HMB 40), or 80 (HMB 80) µmol HMB·kg body weight-1·d-1. Cell replication was assessed from nuclear incorporation of BrdU in the longissimus dorsi (LD) muscle and jejunum crypt cells. Protein synthesis rates in LD, gastrocnemius, rhomboideus, and diaphragm muscles, lung, and brain were greater in HMB 80 and HP, and in brain were greater in HMB 40, compared to LP and F groups. Formation of the eIF4E·eIF4G complex and S6K1 and 4EBP1 phosphorylation in LD, gastrocnemius, and rhomboideus muscles were greater in HMB 80 and HP than in LP and F groups. Phosphorylation of eIF2α and eEF2 and expression of SNAT2, LAT1, MURF1, atrogin-1, and LC3-II were unchanged. Numbers of BrdU positive myonuclei in the LD were greater in HMB 80 and HP than in LP and F groups; there were no differences in jejunum. The results suggest that enteral supplementation with HMB increases skeletal muscle protein anabolism in neonates by stimulation of protein synthesis and satellite cell proliferation.

    May 03, 2016   doi: 10.1152/ajpendo.00520.2015   open full text
  • Metabolic activity of brown, "beige" and white adipose tissues in response to chronic adrenergic stimulation in male mice.
    Labbe, S. M., Caron, A., Chechi, K., Laplante, M., Lecomte, R., Richard, D.
    AJP Endocrinology and Metabolism. May 03, 2016

    Classical brown adipocytes such as those found in interscapular brown adipose tissue (iBAT) represent energy-burning cells, which have been postulated to play a pivotal role in energy metabolism. Brown adipocytes can also be found in white adipose tissue (WAT) depots (e.g. inguinal WAT - iWAT) following adrenergic stimulation and they have been referred to as "beige" adipocytes. Whether the presence of these adipocytes, which gives iWAT a "beige" appearance, can confer a white depot with some thermogenic activity remains to be seen. In consequence, we designed the present study to investigate the metabolic activity of iBAT, iWAT and epididymal white depots in mice. Mice were either (i) kept at thermoneutrality (30ºC), (ii) kept at 30ºC and treated daily for 14 days with an adrenergic agonist (CL) or (iii) housed at 10ºC for 14 days. Metabolic activity was assessed using PET imaging with 18F-fluorodeoxyglucose (glucose uptake), 18F-fluoro-thia-heptadecanoic acid (fatty acid uptake) and 11C-acetate (oxidative activity). In each group, substrate uptakes and oxidative activity were measured in anaesthetized mice in response to acute CL. Our results revealed iBAT as a major site of metabolic activity, which exhibited enhanced glucose and NEFA uptakes and oxidative activity in response to chronic cold and CL. On the other hand, "beige" adipose tissue failed to exhibit appreciable increased in oxidative activity in response to chronic cold and CL. Altogether, our results suggest that the contribution of "beige" fat to acute-CL-induced metabolic activity is low compared to that of iBAT, even after sustained adrenergic stimulation.

    May 03, 2016   doi: 10.1152/ajpendo.00545.2015   open full text
  • Gut hormone secretion, gastric emptying and glycemic responses to erythritol and xylitol in lean and obese subjects.
    Woelnerhanssen, B. K., Cajacob, L., Keller, N., Doody, A., Drewe, J., Rehfeld, J. F., Peterli, R., Beglinger, C., Meyer-Gerspach, A. C.
    AJP Endocrinology and Metabolism. April 26, 2016

    With the increasing prevalence of obesity and a possible association with increasing sucrose consumption, non-nutritive sweeteners are gaining popularity. Given that some studies indicate that artificial sweeteners might have adverse effects, and alternative solutions are sought. Xylitol and erythritol have been known for a long time and their beneficial effects on caries prevention and potential health benefits in diabetic patients have been demonstrated in several studies. Glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK) are released from the gut in response to food intake, promote satiation, reduce gastric emptying (GE) and modulate glucose homeostasis. While glucose ingestion stimulates sweet taste receptors in the gut, and leads to incretin and gastrointestinal hormone release, the effect of xylitol and erythritol have not been well studied. Ten lean and 10 obese volunteers were given 75g glucose, 50g xylitol or 75g erythritol in 300mL water or placebo (water) by a nasogastric tube. We examined plasma glucose, insulin, active GLP-1, CCK, and GE with a 13C-sodium acetate breath test and assessed subjective feelings of satiation. Xylitol and erythritol lead to a marked increase in CCK and GLP-1, while insulin and plasma glucose are not (erythritol) or only slightly (xylitol) affected. Both xylitol and erythritol induce a significant retardation in GE. Subjective feelings of appetite are not significantly different after carbohydrate intake compared to placebo. In conclusion, acute ingestion of erythritol and xylitol stimulates gut hormone release and slows down gastric emptying, while there is no or only little effect on insulin release.

    April 26, 2016   doi: 10.1152/ajpendo.00037.2016   open full text
  • Involvement of a pro-apoptotic gene BBC3 in islet injury mediated by cold preservation and re-warming.
    Omori, K., Kobayashi, E., Komatsu, H., Rawson, J., Agrawal, G., Parimi, M., Oancea, A. R., Valiente, L., Ferreri, K., Al-Abdullah, I. H., Kandeel, F., Takahashi, M., Mullen, Y.
    AJP Endocrinology and Metabolism. April 26, 2016

    Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and re-warming-induced injury by interrogating a pro-apoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3 knockout (KO) mice, and 3) cold preservation and re-warming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during re-warming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved post-preservation mitochondrial viability. Cold preservation resulted in decreased post-isolation islet yield in both wild type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3 KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3 KO islets from cold-preserved pancreata showed reduced HMGB1 (High-mobility Group Box 1 protein) expression and decreased levels of 4-hydroxynonenal (4HNE) protein adducts, indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was up-regulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after re-warming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/re-warming-mediated islet injury, possibly through modulating HMGB1-and oxidative stress-mediated injury to islets.

    April 26, 2016   doi: 10.1152/ajpendo.00441.2015   open full text
  • Exposure to environmentally persistent free radicals during gestation lowers energy expenditure and impairs skeletal muscle mitochondrial function in adult mice.
    Stephenson, E. J., Ragauskas, A., Jaligama, S., Redd, J. R., Parvathareddy, J., Peloquin, M. J., Saravia, J., Han, J. C., Cormier, S. A., Bridges, D.
    AJP Endocrinology and Metabolism. April 26, 2016

    We have investigated the effects of in utero exposure to Environmentally Persistent Free Radicals (EPFR's) on growth, metabolism, energy utilization and skeletal muscle mitochondria in a mouse model of diet-induced obesity. Pregnant mice were treated with laboratory-generated combustion derived particular matter (MCP230). The adult offspring were placed on a high fat diet for 12 weeks, after which we observed a 9.8% increase in their body weight. The increase in body size observed in the MCP230-exposed mice was not associated with increases in food intake, but was associated with a reduction in physical activity and lower energy expenditure. The reduced energy expenditure in mice indirectly exposed to MCP230 was associated with reductions in skeletal muscle mitochondrial DNA copy number, lower mRNA levels of electron transport genes and reduced citrate synthase activity. Up-regulation of key genes involved in ameliorating oxidative stress was also observed in the muscle of MCP230-exposed mice. These findings suggest that gestational exposure to MCP230 leads to a reduction in energy expenditure, at least in part, through alterations to mitochondrial metabolism in the skeletal muscle.

    April 26, 2016   doi: 10.1152/ajpendo.00521.2015   open full text
  • Diacylglycerol Kinase {delta} Regulates AMPK Signaling, Lipid Metabolism and Skeletal Muscle Energetics.
    Jiang, L. Q., De Castro Barbosa, T., Massart, J., Deshmukh, A. S., Lofgren, L., Duque-Guimaraes, D. E., Ozilgen, A., Osler, M. E., Chibalin, A. V., Zierath, J. R.
    AJP Endocrinology and Metabolism. November 03, 2015

    Decrease of AMPK-related signal transduction and insufficient lipid oxidation contributes to the pathogenesis of obesity and type 2 diabetes. Previously, we identified that diacylglycerol kinase (DGK, an enzyme involved in triglyceride biosynthesis, is reduced in skeletal muscle from type 2 diabetic patients. Here we test the hypothesis that DGK plays a role in maintaining appropriate AMPK action in skeletal muscle and energetic aspects of contraction. Voluntary running activity was reduced in DGK+/- mice, but glycogen content and mitochondrial markers were unaltered, suggesting DGK deficiency affects skeletal muscle energetics, but not mitochondrial protein abundance. We next determined the role of DGK in AMPK-related signal transduction and lipid metabolism in isolated skeletal muscle. AMPK activation and signaling was reduced in DGK+/- mice, concomitant with impaired lipid oxidation and elevated incorporation of free fatty acids into triglycerides. Strikingly, DGK deficiency impaired work performance as evident by altered force-production and relaxation dynamics in response to repeated contractions. In conclusion, DGK deficiency impairs AMPK signaling and lipid metabolism, thereby highlighting the deleterious role of excessive lipid metabolites in the development of peripheral insulin resistance and type 2 diabetes pathogenesis. DGK deficiency also influences skeletal muscle energetics, which may lead to low physical activity levels in type 2 diabetes.

    November 03, 2015   doi: 10.1152/ajpendo.00209.2015   open full text
  • Stromal cell-derived factor-1 mediates changes of bone marrow stem cells during bone repair process.
    Okada, K., Kawao, N., Yano, M., Tamura, Y., Kurashimo, S., Okumoto, K., Kojima, K., Kaji, H.
    AJP Endocrinology and Metabolism. November 03, 2015

    Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The numbers of HSCs and MSCs in the bone marrow of mice with damaged femurs were significantly lower and higher, respectively, than the numbers of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Conversely, this effect was not observed within the spleens of mice after bone damage. Meanwhile, both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease and increase in HSC and MSC populations, respectively, within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.

    November 03, 2015   doi: 10.1152/ajpendo.00253.2015   open full text
  • Evidence for hypothalamic ketone bodies sensing: impact on food intake and peripheral metabolic responses in mice.
    Carneiro, L., Geller, S., Fioramonti, X., Hebert, A., Repond, C., Leloup, C., Pellerin, L.
    AJP Endocrinology and Metabolism. November 03, 2015

    Monocarboxylates have been implicated in the control of energy homeostasis. Among them, the putative role of ketone bodies produced notably during high fat diet (HFD) has not been thoroughly explored. In this study, we aimed to determine the impact of a specific rise in cerebral ketone bodies on food intake and energy homeostasis regulation. A carotid infusion of ketone bodies was performed on mice to stimulate sensitive brain areas during 6 or 12 hours. At each time point, food intake and different markers of energy homeostasis were analyzed to reveal the consequences of cerebral increase in ketone bodies level detection. First, an increase in food intake appeared over a 12-hour period of brain ketone bodies perfusion. This stimulated food intake was associated with an increased expression of the hypothalamic neuropeptides NPY and AgRP as well as of phosphorylated AMPK and is due to ketone bodies sensed by the brain as blood ketone bodies levels did not change at that time. In parallel, gluconeogenesis and insulin sensitivity were transiently altered. Indeed, a dysregulation of glucose production and insulin secretion was observed after 6 hours of ketone bodies perfusion which reversed to normal at 12 hours of perfusion. Altogether, these results suggest that an increase in brain ketone bodies concentration leads to hyperphagia and a transient perturbation of peripheral metabolic homeostasis.

    November 03, 2015   doi: 10.1152/ajpendo.00282.2015   open full text
  • Enhanced Insulin Sensitivity by Adipose Tissue Browning Alters Islet Morphology and Hormone Secretion in Response to Autonomic Nervous Activation in Female Mice.
    Omar, B. A., Kvist-Reimer, M., Enerback, S., Ahren, B.
    AJP Endocrinology and Metabolism. November 03, 2015

    Insulin resistance results in compensatory increase in insulin secretion to maintain normoglycemia. Conversely, high insulin sensitivity results in reduced insulin secretion to prevent hypoglycemia. The mechanisms for this inverse adaptation are not well understood. We utilized highly insulin sensitive mice, due to adipocyte specific overexpression of the FOXC2 transcription factor, to study mechanisms of the reversed islet adaptation to increased insulin sensitivity. We found that Foxc2TG mice responded to mild hyperglycemia with reduced insulin secretion compared to wild type mice, however when severe hyperglycemia was induced, Foxc2TG mice demonstrated insulin secretion equal to or greater than that of wild type mice. In response to autonomic nervous activation the acute suppression of insulin seen in wild-type mice was absent in Foxc2TG mice suggesting impaired adrenergic signaling in the islet. Basal glucagon was increased in Foxc2TG mice but they displayed severely impaired glucagon responses to cholinergic and autonomic nervous stimuli. These data suggest that the autonomic nerves contribute to the islet adaptation to high insulin sensitivity which is compatible with a neuro-adipo regulation of islet function being instrumental for maintaining glucose regulation.

    November 03, 2015   doi: 10.1152/ajpendo.00296.2015   open full text
  • Short-term sleep deprivation with nocturnal light exposure alters time-dependent glucagon-like peptide-1 and insulin secretion in male volunteers.
    Gil-Lozano, M., Hunter, P. M., Behan, L.-A., Gladanac, B., Casper, R. F., Brubaker, P. L.
    AJP Endocrinology and Metabolism. November 03, 2015

    The intestinal L-cell is the principal source of glucagon-like peptide-1 (GLP-1), a major determinant of insulin release. As GLP-1 secretion is regulated in a circadian manner in rodents, we investigated whether the activity of the human L-cell is also time-sensitive. Rhythmic fluctuations in the mRNA levels of canonical clock genes were found in the human NCI-H716 L-cell model, which also showed a time-dependent pattern in their response to well-established secretagogues. A diurnal variation in GLP-1 responses to identical meals (850 kcal), served 12 h apart in the normal dark (23:00) and light (11:00) periods, was also observed in male volunteers maintained under standard sleep and light conditions. These findings suggest the existence of a daily pattern of activity in the human L-cell. Moreover, we separately tested the short-term effects of sleep-deprivation and nocturnal light exposure on basal and postprandial GLP-1, insulin and glucose levels in the same volunteers. Sleep-deprivation with nocturnal light exposure disrupted the melatonin and cortisol profiles, and increased insulin resistance. Moreover, it also induced profound derangements in GLP-1 and insulin responses, such that postprandial GLP-1 and insulin levels were markedly elevated and the normal variation in GLP-1 responses was abrogated. These alterations were not observed in sleep-deprived participants maintained under dark conditions, indicating a direct effect of light on the mechanisms that regulate glucose homeostasis. Accordingly, the metabolic abnormalities known to occur in shift-workers may be related to the effects of irregular light-dark cycles on these glucoregulatory pathways.

    November 03, 2015   doi: 10.1152/ajpendo.00298.2015   open full text
  • Equine hyperinsulinemia: investigation of the enteroinsular axis during insulin dysregulation.
    de Laat, M. A., McGree, J. M., Sillence, M. N.
    AJP Endocrinology and Metabolism. November 03, 2015

    Compared to other species insulin dysregulation in equids is poorly understood. Hyperinsulinemia causes laminitis, a significant and often lethal disease affecting the pedal bone/hoof wall attachment site. Until recently, hyperinsulinemia has been considered a counter-regulatory response to insulin resistance (IR), but there is growing evidence to support a gastrointestinal etiology. Incretin hormones released from the proximal intestine, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide, augment insulin secretion in several species, but require investigation in horses. This study investigated peripheral and gut-derived factors impacting insulin secretion by comparing the response to intravenous (IV) and oral D-glucose. Oral and IV tests were performed in 22 ponies previously shown to be insulin dysregulated, of which only 15 were classified as IR (IV test). In a more detailed study, nine different ponies received four treatments: D-glucose orally, D-glucose IV, oats and Workhorse-mix. Insulin, glucose and incretin concentrations were measured before and after each treatment. All nine ponies showed similar IV responses, but five were markedly hyper-responsive to oral D-glucose and four were not. Insulin responsiveness to oral D-glucose was strongly associated with blood glucose concentrations and oral glucose bioavailability, presumably driven by glucose absorption/distribution, as there was no difference in glucose clearance rates. Insulin was also positively associated with active GLP-1 following D-glucose and grain. This study has confirmed a functional enteroinsular axis in ponies which likely contributes to insulin dysregulation that may predispose them to laminitis. Further, IV tests for IR are not reliable predictors of the oral response to dietary non-structural carbohydrate.

    November 03, 2015   doi: 10.1152/ajpendo.00362.2015   open full text
  • The anabolic response to a meal containing different amounts of protein is not limited by the maximal stimulation of protein synthesis in healthy young adults.
    Kim, I.-Y., Schutzler, S., Schrader, A., Spencer, H. J., Azhar, G., Ferrando, A. A., Wolfe, R. R.
    AJP Endocrinology and Metabolism. November 03, 2015

    We have determined whole body protein kinetics i.e., protein synthesis (PS), breakdown (PB), and net balance (NB) in human subjects in the fasted state and following ingestion of ~40g (moderate protein, or MP) that has been reported to maximized the protein synthetic response or ~70g (higher protein, HP) protein, more representative of the amount of protein in the dinner of an average American diet. Twenty three healthy young men who had performed prior resistance exercise (X-MP or X-HP) or time-matched resting (R-MP or R-HP) were studied during a primed continuous infusion of L-[2H5]phenylalanine and L-[2H2]tyrosine. Subjects were randomly assigned into an exercise (X, n=12) or resting (R, n=11) group, and each group was studied at the two levels of dietary protein intake in random order. PS, PB, and NB were expressed as increases above the basal, fasting values (mg/kg LBM/min). Exercise did not significantly affect protein kinetics and blood chemistry. Feeding resulted in positive NB at both levels of protein intake: NB was greater in response to the meal containing HP (p<0.00001). The greater NB with HP was achieved primarily through a greater reduction in PB and to a lesser extent stimulation of protein synthesis (for all, p<0.0001). HP resulted in greater plasma EAA responses (p<0.01) vs. MP, with no differences in insulin and glucose responses. In conclusion, whole body net protein balance improves with greater protein intake above that previously suggested to maximally stimulating muscle protein synthesis because of a simultaneous reduction in protein breakdown.

    November 03, 2015   doi: 10.1152/ajpendo.00365.2015   open full text
  • The Impact of a Human IGF-II Analogue ([Leu27]IGF-II) on Fetal Growth in a Mouse Model of Fetal Growth Restriction.
    Charnock, J. C., Dilworth, M. R., Aplin, J. D., Sibley, C. P., Westwood, M., Crocker, I. P.
    AJP Endocrinology and Metabolism. November 03, 2015

    Enhancing placental insulin-like growth factor(IGF) availability appears an attractive strategy for improving outcomes in fetal growth restriction(FGR). Our approach was the use of [Leu27]IGFII, a human IGF-II analogue that binds the IGF-II clearance receptor, IGF2R, in FGR mice. We hypothesised that the impact of [Leu27]IGFII infusion in C57BL/6J(wild-type,WT) and endothelial nitric oxide synthase knockout(eNOS-/-,FGR) mice would be to enhance fetal growth and investigated this from mid to late gestation. 1mg/kg/day [Leu27]IGFII was delivered via a subcutaneous mini-osmotic pump from E12.5-E18.5. Fetal and placental weights, recorded at E18.5, were used to generate frequency distribution curves; fetuses <5th centile were deemed growth-restricted. Placentas were harvested for immunohistochemical analysis of the IGF system, and maternal serum collected for measurement of exogenously administered IGF-II. In WT pregnancies, [Leu27]IGFII treatment halved the number of FGR fetuses, reduced fetal(p=0.028) and placental weight variations(p=0.0032), and increased numbers of pups close to the mean fetal weight(131vs112 pups within 1 SD). Mixed-models analysis confirmed litter size to be negatively correlated with fetal and placental weight, and showed that Leu27[IGFII] preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional 14CMeAIB transfer per g placenta(System A amino acid transporter activity) was inversely correlated with fetal weight in Leu27IGFII-treated WT animals(p<0.01). In eNOS-/- mice, [Leu27]IGFII reduced the number of FGR fetuses(1vs5 in the untreated group). The observed reduction in FGR pup numbers in both C57 and eNOS-/- litters suggests the use of this analogue as a means of standardising and rescuing fetal growth, preferentially in the smallest offspring.

    November 03, 2015   doi: 10.1152/ajpendo.00379.2015   open full text
  • Advances in High Density Lipoprotein Physiology: surprises, overturns and promises.
    Constantinou, C., Karavia, E. A., Xepapadaki, E., Petropoulou, P.-I., Papakosta, E., Karavyraki, M., Zvintzou, E., Theodoropoulos, V., Filou, S., Hatziri, A., Kalogeropoulou, C., Panayiotakopoulos, G., Kypreos, K. E.
    AJP Endocrinology and Metabolism. November 03, 2015

    Emerging evidence strongly supports that changes in HDL metabolic pathway that result into changes in HDL proteome and function, appear to have a causative impact on a number of metabolic disorders. Here, we provide a critical review of the most recent and novel findings correlating HDL properties and functionality with various pathophysiological processes and disease states, such as obesity, type 2 diabetes mellitus, nonalcoholic fatty liver disease, inflammation and sepsis, bone and obstructive pulmonary diseases, and brain disorders.

    November 03, 2015   doi: 10.1152/ajpendo.00429.2015   open full text
  • Leptin acts in the brain to influence hypoglycemic counterregulation: Disparate effects of acute and recurrent hypoglycemia on glucagon release.
    Reno, C. M., Ding, Y., Sherwin, R.
    AJP Endocrinology and Metabolism. October 27, 2015

    Leptin has been shown to diminish hyperglycemia via reduced glucagon secretion, although it can also enhance sympathoadrenal responses. Whether leptin can also inhibit glucagon secretion during insulin-induced hypoglycemia or increase epinephrine during acute or recurrent hypoglycemia has not, however, been examined. To test if leptin acts in the brain to influence counterregulation, hyperinsulinemic/hypoglycemic (~45 mg/dl) clamps were performed on rats exposed to or not exposed to recurrent hypoglycemia (3 days, ~40 mg/dl). Intracerebroventricular artificial cerebral spinal fluid or leptin were infused during the clamp. During acute hypoglycemia, leptin decreased glucagon responses by 51%, but increased epinephrine and norepinephrine by 24% and 48%, respectively. After recurrent hypoglycemia, basal plasma leptin levels were undetectable. Subsequent brain leptin infusion during hypoglycemia paradoxically increased glucagon by 45% as well as epinephrine by 19%. In conclusion, leptin acts within the brain to diminish glucagon secretion during acute hypoglycemia, but increases epinephrine potentially limiting its detrimental effects during hypoglycemia. Exposure to recurrent hypoglycemia markedly suppresses plasma leptin, whereas exogenous brain leptin delivery enhances both glucagon and epinephrine release to subsequent hypoglycemia. These data suggest that recurrent hypoglycemia may diminish counterregulatory responses in part by reducing brain leptin action.

    October 27, 2015   doi: 10.1152/ajpendo.00361.2015   open full text
  • A Systems Based Investigation into Vitamin D and Skeletal Muscle Repair, Regeneration and Hypertrophy.
    Owens, D. J., Sharples, A. P., Polydorou, I., Alwan, N., Donovan, T. F., Tang, J., Cooper, R. G., Fraser, W. D., Morton, J. P., Stewart, C., Close, G. L.
    AJP Endocrinology and Metabolism. October 27, 2015

    Skeletal muscle is a direct target for Vitamin D. Observational studies suggest that 25[OH]D correlates with recovery of skeletal muscle following eccentric contractions in humans and crush injury in rats. However, a definitive association is yet to be established. In order to address this gap in knowledge in relation to damage repair, a randomised, placebo-controlled trial was performed in twenty males with low serum 25[OH]D (45 ± 25 nmol.L-1). Prior to and following 6-weeks of supplemental Vitamin D3 (4,000 or placebo (50 mg cellulose), participants performed 20x10 damaging eccentric contractions of the knee extensors with peak torque measured over the following 7 days of recovery. Parallel experimentation using isolated human skeletal muscle derived myoblast cells from biopsies of 14 males with insufficient serum 25[OH]D (37 ± 11 nmol.L-1) were subjected to mechanical wound injury, which enabled corresponding in vitro studies of muscle repair, regeneration and hypertrophy in the presence and absence of 10 nmol or 100 nmol 1α,25[OH]2D3. Supplemental Vitamin D3 increased serum 25[OH]D and improved recovery of peak torque at 48 hours and 7 days post-exercise. In vitro, 10 nmol 1α,25[OH]2D3 improved muscle cell migration dynamics and resulted in improved myotube fusion/differentiation at the biochemical, morphological and molecular level together with increased myotube hypertrophy at 7 and 10 days post-damage. Together, these preliminary data are the first to characterise a role for Vitamin D in human skeletal muscle regeneration and suggest that maintaining serum 25[OH]D may be beneficial for enhancing reparative processes and potentially for facilitating subsequent hypertrophy.

    October 27, 2015   doi: 10.1152/ajpendo.00375.2015   open full text
  • Increased intramyocellular lipid/impaired insulin sensitivity is associated with altered lipid metabolic genes in muscle of high responders to a high fat diet.
    Kakehi, S., Tamura, Y., Takeno, K., Sakurai, Y., Kawaguchi, M., Watanabe, T., Funayama, T., Sato, F., Ikeda, S.-i., Kanazawa, A., Fujitani, Y., Kawamori, R., Watada, H.
    AJP Endocrinology and Metabolism. October 20, 2015

    The accumulation of intramyocellular lipid (IMCL) is recognized as an important determinant of insulin resistance, and is increased by a high-fat diet (HFD). However, the effects of HFD on IMCL and insulin sensitivity are highly variable. The aim of this study was to identify the genes in muscle that are related to this inter-individual variation. Fifty healthy men were recruited for this study. Before and after HFD for 3 days, IMCL levels in the tibialis anterior were measured by 1H-magnetic resonance spectroscopy, and peripheral insulin sensitivity was evaluated by glucose infusion rate (GIR) during the euglycemic-hyperinsulinemic clamp. Subjects who showed a large increase in IMCL and a large decrease in GIR by HFD were classified as high-responders (HRs), and subjects who showed a small increase in IMCL and a small decrease in GIR were classified as low-responders (LRs). In 5 subjects from each group, the gene expression profile of the vastus lateralis muscle was analyzed by DNA microarray analyses. Before HFD, gene expression profiles related to lipid metabolism were comparable between the 2 groups. Gene Set Enrichment Analysis demonstrated that 5 gene sets related to lipid metabolism were up-regulated by HFD in the HR group, but not in the LR group. Changes in gene expression patterns were confirmed by qRT-PCR using more samples (LR: n = 9; HR: n = 11). These results suggest that IMCL accumulation/impaired insulin sensitivity after HFD is closely associated with changes in the expression of genes related to lipid metabolism in muscle.

    October 20, 2015   doi: 10.1152/ajpendo.00220.2015   open full text
  • Disruption of REDD1 gene ameliorates sepsis-induced decrease in mTORC1 signaling but has divergent effects on proteolytic signaling in skeletal muscle.
    Steiner, J. L., Crowell, K. T., Kimball, S. R., Lang, C. H.
    AJP Endocrinology and Metabolism. October 20, 2015

    Sepsis-induced skeletal muscle atrophy and weakness is due in part to decreased mTORC1-mediated protein synthesis and increased proteolysis via the autophagy-lysosomal system and ubiquitin-proteasome pathway. The REDD (Regulated in development and DNA damage)-1 protein is increased in sepsis and can negatively regulate mTORC1 activity. However, the contribution of REDD1 to the sepsis-induced change in muscle protein synthesis and degradation has not been determined. Sepsis was produced by cecal ligation and puncture in female REDD1-/- or wildtype (WT) mice and endpoints were assessed 24 h later in gastrocnemius; time-matched, pair-fed controls of each genotype were included. Sepsis increased REDD1 protein 300% in WT mice while REDD1 was absent in REDD1-/- muscle. Sepsis decreased protein synthesis and phosphorylation of downstream targets of mTORC1 (S6K1 T389, rpS6 S240/244, 4E-BP1 S65) in WT but not REDD1-/- mice. However, Akt and PRAS40 phosphorylation was suppressed in both sham and septic muscle from REDD1-/- mice, despite unaltered PDK1, PP2A or TSC2 expression. Sepsis increased autophagy as indicated by decreased ULK1 S757 phosphorylation and p62 abundance, and increased LC3B-II/I in WT mice, while these changes were absent in septic REDD1-/- mice. Conversely, REDD1 deletion did not prevent the sepsis-induced decrease in IGF-I mRNA or the concomitant increase in IL-6, TNF-α, MuRF1 and atrogin1 mRNA expression. Lastly, 5 day survival in a separate set of septic mice did not differ between WT and REDD1-/- mice. These data highlight the central role of REDD1 in regulating both protein synthesis and autophagy in skeletal muscle during sepsis.

    October 20, 2015   doi: 10.1152/ajpendo.00264.2015   open full text
  • 2-Methoxyestradiol Blocks the RhoA/ROCK1 Pathway in Human Aortic Smooth Muscle Cells.
    Rigassi, L., Bozzolo, F. B., Lucchinetti, E., Zaugg, M., Fingerle, J., Rosselli, M., Imthurn, B., Jackson, E. K., Dubey, R. K.
    AJP Endocrinology and Metabolism. October 20, 2015

    2-Methoxyestradiol (2-ME), a metabolite of estradiol with little affinity for estrogen receptors, inhibits proliferation of vascular smooth muscle cells; however the molecular mechanisms underlying this effect are incompletely understood. Our previous work shows that 2-ME inhibits initiation (blocks phosphorylation of ERK and Akt) and progression (reduces cyclin expression and increases expression of cyclin inhibitors) of the mitogenic pathway and interferes with mitosis (disrupts tubulin organization). Because the RhoA/ROCK1 pathway (RhoA -> ROCK1 -> myosin phosphatase targeting subunit -> myosin light chain) is involved in cytokinesis, here we test the concept that 2-ME also blocks the RhoA/ROCK1 pathway. Because of the potential importance of 2-ME for preventing/treating vascular diseases, experiments were conducted in female human aortic vascular smooth muscles (HASMCs). Microarray transcriptional profiling suggested an effect of 2-ME on the RhoA/ROCK1 pathway. Indeed 2-ME blocked mitogen-induced GTP-bound RhoABC expression and membrane bound RhoA, suggesting interference with the activation of RhoA. 2-ME also reduced ROCK1 expression, suggesting reduced production of the primary downstream signaling kinase of the RhoA pathway. Moreover, 2-ME inhibited RhoA/ROCK1 pathway downstream signaling including phosphorylated myosin phosphatase targeting subunit and myosin light chain; the ROCK1 inhibitor H-1152 mimicked these effects of 2-ME. Both 2-ME and H-1152 blocked cytokinesis. 2-ME also reduced the expression of tissue factor, yet another downstream signaling component of the RhoA/ROCK1 pathway. We conclude that 2-ME inhibits the pathway RhoA -> ROCK1 -> myosin phosphatase targeting subunit -> myosin light chain; and this likely contributes to the reduced cytokinesis in 2-ME treated HASMCs.

    October 20, 2015   doi: 10.1152/ajpendo.00267.2015   open full text
  • Bone microstructural defects and osteopenia in hemizygous {beta}IVSII-654 knockin thalassemic mice: sex-dependent changes in bone density and osteoclast function.
    Thongchote, K., Svasti, S., Teerapornpuntakit, J., Suntornsaratoon, P., Krishnamra, N., Charoenphandhu, N.
    AJP Endocrinology and Metabolism. October 20, 2015

    β-thalassemia, a hereditary anemic disorder, is often associated with skeletal complications that can be found in both male and female. The present study aimed to investigate the age- and sex-dependent changes in bone mineral density (BMD) and trabecular microstructure in βIVSII-654 knockin thalassemic mice. Dual energy X-ray absorptiometry (DXA) and computer-assisted bone histomorphometry was employed to investigate temporal changes in BMD and histomorphometric parameters in male and female βIVSII-654 knockin mouse model of human β-thalassemia, in which impaired splicing of β-globin transcript was caused by hemizygous C->T mutation at nucleotide 654 of the intron 2. Young growing βIVSII-654 mice (1 month old) manifested shorter bone length and lower BMD than their wild-type littermates, indicating possible growth retardation and osteopenia, the latter of which persisted until 8 months of age (adult mice). Interestingly, two-way analysis of variance suggested an interaction between gender and βIVSII-654 genotype, i.e., more severe osteopenia in adult female mice. Bone histomorphometry further suggested that low trabecular bone volume in male βIVSII-654 mice, particularly during a growing period (1-2 months), was primarily due to suppression of bone formation, whereas both low bone formation rate and a marked increase in osteoclast surface were observed in female βIVSII-654 mice. In conclusion, osteopenia and trabecular microstructural defects were present in both male and female βIVSII-654 knockin thalassemic mice, but the severity, disease progression and cellular mechanism differed between the two genders.

    October 20, 2015   doi: 10.1152/ajpendo.00329.2015   open full text
  • Diabetes prevalence in NZO females depends on estrogen action on liver fat content.
    Lubura, M., Hesse, D., Kramer, M., Hallahan, N., Schupp, M., Loeffelholz, C. v., Kriebel, J., Rudovich, N. N., Pfeiffer, A. F., John, C., Scheja, L., Heeren, J., Koliaki, C., Roden, M., Schurmann, A.
    AJP Endocrinology and Metabolism. October 20, 2015

    In humans and rodents risk of metabolic syndrome is sexually dimorphic, with an increased incidence in males. Additionally, the protective role of female gonadal hormones is ostensible as prevalence of type 2 diabetes mellitus (T2DM) increases after menopause. Here, we investigated the influence of estrogen (E2) on the onset of T2DM in female New Zealand Obese (NZO) mice. Diabetes prevalence (defined as blood glucose levels >16.6 mmol/l) of NZO females on high-fat diet (60kcal% fat) at week 22 was 43%. This was markedly dependent on liver fat content in week 10, as detected by computed tomography. Only mice with a liver fat content >9% in week 10 plus glucose levels >10 mmol/l in week 9 developed hyperglycaemia by week 22. In addition, at 11 weeks diacylglycerols were elevated in livers of diabetes-prone mice compared to controls. Hepatic expression profiles obtained from diabetes-prone and -resistant mice at 11 weeks revealed increased abundance of two transcripts in diabetes-prone mice: Mogat1 which catalyzes the synthesis of diacylglycerols from monoacylglycerol and fatty acyl-CoA and the fatty acid transporter Cd36. E2-treatment of diabetes-prone mice for 10 weeks prevented any further increase in liver fat content, reduced diacylglycerols and the abundance of Mogat1 and Cd36 leading to a reduction of diabetes prevalence and an improved glucose tolerance compared to untreated mice. Our data indicates that early elevation of hepatic Cd36 and Mogat1 associates with increased production and accumulation of triglycerides and diacylglycerols, presumably resulting in reduced hepatic insulin sensitivity and leading to later onset of T2DM.

    October 20, 2015   doi: 10.1152/ajpendo.00338.2015   open full text
  • Gastric Inhibitory Polypeptide Immunoneutralization Attenuates Development of Obesity in Mice.
    Boylan, M. O., Glazebrook, P. A., Tatalovic, M., Wolfe, M. M.
    AJP Endocrinology and Metabolism. October 20, 2015

    Background & Aims: Previous reports have suggested that the abrogation of gastric inhibitory polypeptide (GIP) signaling could be exploited to prevent and treat obesity and obesity-related disorders in humans. This study was designed to determine whether immunoneutralization of GIP using a newly developed specific mAb would prevent the development of obesity. Methods: A specific mAb directed against the carboxy-terminus of mouse GIP was identified, and its effects on the insulin response to oral and to IP glucose and on weight gain were evaluated. Results: Administration of mAb (30 mg/kg body weight, BW) to mice attenuated the insulin response to oral glucose by 70% and completely eliminated the response to IP glucose co-administered with human GIP. Nine week (wk)-old C57BBL/6 mice injected with GIP mAbs (60 mg/kg BW/wk) for 17 wk gained 46.5% less weight than control mice fed an identical high-fat diet (P=0.00000007). When corrected for BW, no difference in the quantity of food consumed was detected between the two treatment groups. Furthermore, MRI demonstrated that subcutaneous, omental, and hepatic fat were 1.97-, 3.46- and 2.15-fold, respectively, lower in mAb-treated animals compared to controls. Moreover, serum insulin, leptin, total cholesterol (TC), LDL, and triglycerides (TG) were significantly reduced, while the HDL:TC ratio was 1.25-fold higher in treated animals compared to controls. Conclusions: These studies support the hypothesis that a reduction in GIP signaling using a GIP-neutralizing mAb might provide a useful method for the treatment and prevention of obesity and related disorders.

    October 20, 2015   doi: 10.1152/ajpendo.00345.2015   open full text
  • Multifaceted interplay among mediators and regulators of intestinal glucose absorption: Potential impacts on diabetes research and treatment.
    Chan, L. K., Leung, P. S.
    AJP Endocrinology and Metabolism. October 20, 2015

    Glucose is the prominent molecule that characterizes diabetes and, like the vast majority of nutrients in our diet, it is absorbed and enters the bloodstream directly through the small intestine, and hence small intestine physiology impacts blood glucose levels directly. Accordingly, intestinal regulatory modulators represent a promising avenue through which diabetic blood glucose levels might be moderated clinically. Despite the critical role of small intestine in blood glucose homeostasis, most physiological diabetes research has focused on other organs, such as the pancreas, kidney, and liver. We contend that an improved understanding of intestinal regulatory mediators may be fundamental for the development of first-line preventive and therapeutic interventions in patients with diabetes and diabetes-related diseases. This review summarizes the major important intestinal regulatory mediators, discusses how they influence intestinal glucose absorption, and suggests possible candidates for future diabetes research and the development of anti-diabetic therapeutic agents.

    October 20, 2015   doi: 10.1152/ajpendo.00373.2015   open full text
  • Ingestion of Glucose or Sucrose Prevents Liver but not Muscle Glycogen Depletion During Prolonged Endurance-type Exercise in Trained Cyclists.
    Gonzalez, J. T., Fuchs, C. J., Smith, F. E., Thelwall, P. E., Taylor, R., Stevenson, E. J., Trenell, M. I., Cermak, N. M., van Loon, L. J. C.
    AJP Endocrinology and Metabolism. October 20, 2015

    Purpose: To define the effect of glucose ingestion compared to sucrose ingestion on liver and muscle glycogen depletion during prolonged endurance-type exercise. Methods: Fourteen cyclists completed two 3-h bouts of cycling at 50% of peak power output while ingesting either glucose or sucrose at a rate of 1.7 g/min (102 g/h). Four cyclists performed an additional third test in which only water was consumed for reference. We employed 13C magnetic resonance spectroscopy to determine liver and muscle glycogen concentrations before and after exercise. Expired breath was sampled during exercise to estimate whole-body substrate use. Results: Following glucose and sucrose ingestion, liver glycogen levels did not show a significant decline following exercise (from 325±168 to 345±205 and 321±177 to 348±170 mmol/L, respectively; P>0.05) with no differences between treatments. Muscle glycogen concentrations declined (from 101±49 to 60±34 and 114±48 to 67±34 mmol/L, respectively; P<0.05), with no differences between treatments. Whole-body carbohydrate utilization was greater with sucrose (2.03±0.43 g/min) vs glucose ingestion (1.66±0.36 g/min; P<0.05). Both liver (from 454±33 to 283±82 mmol/L; P<0.05) and muscle (from 111±46 to 67±31 mmol/L; P<0.01) glycogen concentrations declined during exercise when only water was ingested. Conclusion: Both glucose and sucrose ingestion prevent liver glycogen depletion during prolonged endurance-type exercise. Sucrose ingestion does not preserve liver glycogen concentrations more than glucose ingestion. However, sucrose ingestion does increase whole-body carbohydrate utilization compared to glucose ingestion. This trial was registered at as NCT02110836.

    October 20, 2015   doi: 10.1152/ajpendo.00376.2015   open full text
  • Mechanisms for Greater Insulin-stimulated Glucose Uptake in Normal and Insulin Resistant Skeletal Muscle after Acute Exercise.
    Cartee, G. D.
    AJP Endocrinology and Metabolism. October 20, 2015

    Enhanced skeletal muscle and whole body insulin sensitivity can persist up to 24-48 hours after one exercise session. This review focuses on potential mechanisms for greater post-exercise, insulin-stimulated glucose uptake (ISGU) by muscle in individuals with normal or reduced insulin sensitivity. A model is proposed for the processes underlying this improvement. (1) Triggers are initiating events that activate subsequent (2) memory elements which store information that is relayed to (3) mediators which translate memory into action by controlling (4) an end-effector that directly executes increased insulin-stimulated glucose transport. Several candidates are potential triggers or memory elements, but none has been conclusively verified. Regarding potential mediators, in both normal and insulin resistant individuals, elevated post-exercise ISGU with a physiologic insulin dose coincides with greater Akt Substrate of 160 kDa (AS160) phosphorylation without improved proximal insulin signaling at steps from insulin receptor binding to Akt activity. Causality remains to be established between greater AS160 phosphorylation and improved ISGU. The end-effector for normal individuals is increased GLUT4 translocation, but this remains untested for insulin resistant individuals post-exercise. Following exercise, insulin resistant individuals can attain ISGU values similar to non-exercising healthy controls, but after a comparable exercise protocol performed by both groups, ISGU for the insulin resistant group has been consistently reported to be below post-exercise values for the healthy group. Further research is required to fully understand the mechanisms underlying the improved post-exercise ISGU in individuals with normal or subnormal insulin sensitivity and to explain the disparity between these groups after similar exercise.

    October 20, 2015   doi: 10.1152/ajpendo.00416.2015   open full text
  • Impact of maternal undernutrition around the time of conception on factors regulating hepatic lipid metabolism and microRNAs in singleton and twin fetuses.
    Lie, S., Morrison, J. L., Williams-Wyss, O., Suter, C. M., Humphreys, D. T., Ozanne, S. E., Zhang, S., MacLaughlin, S. M., Kleemann, D. O., Walker, S. K., Roberts, C. T., McMillen, I. C.
    AJP Endocrinology and Metabolism. October 20, 2015

    We have investigated the effects of embryo number and maternal undernutrition imposed either around the time of conception or before implantation on hepatic lipid metabolism in the sheep fetus. We have demonstrated that periconceptional undernutrition and preimplantation undernutrition each resulted in decreased hepatic fatty acid β-oxidation regulators, PGC1α (P<0.05), PDK-2 (P<0.01) and PDK-4 (P<0.01) mRNA expression in singleton and twin fetuses at 135-138 days gestation. In singletons, there was also lower hepatic PDK-4 (P<0.01), CPT-1 (P<0.01), and PKC (P<0.01) protein abundance in the PCUN and PIUN groups, and a lower protein abundance of PDPK-1 (P<0.05) in the PCUN group. Interestingly in twins, the hepatic protein abundance of pAMPK (Ser485) (P<0.01), pPDPK-1 (Ser 41) (P<0.05) and PKC (P<0.05) was higher in the PCUN and PIUN groups, and hepatic PDK-4 (P<0.001) and CPT-1 (P<0.05) protein abundance was also higher in the PIUN twin fetus. We also found that the expression of a number of microRNAs were altered in response to PCUN or PIUN and that there is evidence that these changes may underlie the changes in the protein abundance of key regulators of hepatic fatty acid β-oxidation in the PCUN and PIUN groups. Therefore, embryo number and the timing of maternal undernutrition in early pregnancy have a differential impact on hepatic microRNA expression and on the factors that regulate hepatic fatty acid oxidation and lipid synthesis.

    October 20, 2015   doi: 10.1152/ajpendo.00600.2014   open full text
  • Hydrogen sulfide reduces serum triglyceride by activating liver autophagy via AMPK- mTOR pathway.
    Sun, L., Zhang, S., Yu, C., Pan, Z., Liu, Y., Zhao, J., Wang, X., Yun, F., Zhao, H., Yan, S., Yuan, Y., Wang, D., Ding, X., Li, W., Liu, G., Zhao, X., Liu, Z., Li, Y.
    AJP Endocrinology and Metabolism. October 06, 2015

    Autophagy plays an important role in liver triglyceride (TG) metabolism. Inhibition of autophagy could reduce the clearance of TG in the liver. Hydrogen sulfide (H2S) is a potent stimulator of autophagic flux. Recent studies showed H2S is protective against Hypertriglyceridemia (HTG) and noalcoholic fat liver disease (NAFLD), while the mechanism remains to be explored. Here we test the hypothesis that H2S reduces serum TG level and ameliorates NAFLD through stimulating liver autophagic flux by AMPK-mTOR pathway. The level of serum H2S in patients with HTG was lower than that of control subjects. Sodium hydrosulfide (NaHS, H2S donor) markedly reduced serum TG levels of male C57BL/6 mice fed with high-fat diet (HFD), which was abolished by co-administration of chloroquine (CQ), an inhibitor of autophagic flux. In HFD mice, administration of NaSH increased LC3BII to LC3BI ratio, decreased p62 protein level. Meanwhile, NaSH increased the phosphorylation of AMPK, and thus reduced the phosphorylation of mTOR by western blot study. In cultured LO2 cells, high fat treatment reduced the ratio of LC3BII to LC3BI and the phosphorylation of AMPK, which were reversed by the co-administration of NaSH. Knockdown of AMPK by siRNA in LO2 cells blocked the autophagic enhancing effects of NaSH. The same qualitative effect was observed in AMPKα2-/-mice. These results for the first time demonstrated that H2S could reduce serum TG level and ameliorate NAFLD by activating liver autophagy via AMPK- mTOR pathway.

    October 06, 2015   doi: 10.1152/ajpendo.00294.2015   open full text
  • Acute ingestion of citrulline stimulates nitric oxide synthesis in young and older subjects.
    Kim, I.-Y., Schutzler, S. E., Schrader, A., Spencer, H., Azhar, G., Deutz, N. E. P., Wolfe, R. R.
    AJP Endocrinology and Metabolism. October 06, 2015

    To determine if age-associated vascular dysfunction in older adults with heart failure (HF) is due to insufficient synthesis of nitric oxide (NO), we performed two separate studies: 1) a kinetics study with a stable isotope tracer method to determine in vivo kinetics of NO metabolism and 2) a vascular function study using a plethysmography method to determine reactive hyperemic forearm blood flow (RH-FBF) in older and young adults in the fasted state and in response to citrulline ingestion. In the fasted state, NO synthesis per kg body weight (bw) was approximately 50% lower in older vs. young adults and was related to a decreased rate of appearance of the NO precursor arginine. Citrulline ingestion (3g) stimulated de novo arginine synthesis in both older [6.88±0.83 to 35.40±4.90 μmol/kg bw/h] and to a greater extent in young adults [12.02±1.01 to 66.26±4.79 μmol/kg bw/h]. NO synthesis rate increased correspondingly in older [0.17±0.01 to 2.12±0.36 μmol/kg bw/h] and to a greater extent in young adults [0.36±0.04 to 3.57±0.47 μmol/kg bw/h]. Consistent with the kinetic data, RH-FBF in the fasted state was approximately 40% reduced in older vs. young adults. However, citrulline ingestion (10g) failed to increase RH-FBF in either older or young adults. In conclusion, citrulline ingestion improved impaired NO synthesis in older HF adults, but not RH-FBF, suggesting factors other than NO synthesis play a role in the impaired RH-FBF in older HF adults, and/or it may require a longer duration of supplementation to be effective in improving RH-FBF.

    October 06, 2015   doi: 10.1152/ajpendo.00339.2015   open full text
  • AMPK{alpha} is essential for acute exercise-induced gene responses but not for exercise training-induced adaptations in mouse skeletal muscle.
    Fentz, J., Kjobsted, R., Kristensen, C. M., Hingst, J. R., Birk, J. B., Gudiksen, A., Foretz, M., Schjerling, P., Viollet, B., Pilegaard, H., Wojtaszewski, J. F. P.
    AJP Endocrinology and Metabolism. September 29, 2015

    Exercise training increases skeletal muscle expression of metabolic proteins improving the oxidative capacity. Adaptations in skeletal muscle by pharmacologically induced activation of 5'AMP-activated protein kinase (AMPK) are dependent on the AMPKα2 subunit. We hypothesized that exercise training-induced increases in exercise capacity and expression of metabolic proteins as well as acute exercise-induced gene regulation would be compromised in AMPKα1 and -α2 muscle-specific double knockout (mdKO) mice. An acute bout of exercise increased skeletal muscle mRNA content of cytochrome C oxidase subunit I, glucose transporter 4 and VEGF in an AMPK-dependent manner, while cluster of differentiation 36 and fatty acid transport protein 1 mRNA content increased similarly in AMPKα wild type (WT) and mdKO mice. During four weeks of voluntary running wheel exercise training, the AMPKα mdKO mice ran less than WT. Maximal running speed was lower in AMPKα mdKO than WT mice, but increased similarly in both genotypes with exercise training. Exercise training increased quadriceps protein content of ubiquinol-cytochrome-C reductase core protein 1 (UQCRC1), cytochrome C, hexokinase II, plasma membrane fatty acid binding protein and citrate synthase activity more in AMPKα WT than mdKO muscle. However, analysis of a subgroup of mice matched for running distance revealed that only UQCRC1 protein content increased more in WT than mdKO mice with exercise training. Thus, AMPKα1 and -α2 subunits are important for acute exercise-induced mRNA responses of some genes and may be involved in regulating basal metabolic protein expression, but seem to be less important in exercise training-induced adaptations in metabolic proteins.

    September 29, 2015   doi: 10.1152/ajpendo.00157.2015   open full text
  • FADD dominant negative dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy.
    Wang, F., Weng, H., Quon, M. J., Yu, J., Wang, J.-Y., Hueber, A.-O., Yang, P.
    AJP Endocrinology and Metabolism. September 29, 2015

    Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1alpha (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-Associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome, suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members, mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3, 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress, and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress.

    September 29, 2015   doi: 10.1152/ajpendo.00215.2015   open full text
  • Effects of acute exercise on lipid content and dietary lipid uptake in liver and skeletal muscle of lean and diabetic rats.
    Janssens, S., Jonkers, R. A. M., Groen, A. K., Nicolay, K., van Loon, L. J. C., Prompers, J. J.
    AJP Endocrinology and Metabolism. September 29, 2015

    Insulin resistance is associated with ectopic lipid accumulation. Physical activity improves insulin sensitivity, but the impact of exercise on lipid handling in insulin-resistant tissues remains to be elucidated. The present study characterizes the effects of acute exercise on lipid content and dietary lipid partitioning in liver and skeletal muscle of lean and diabetic rats using magnetic resonance spectroscopy (MRS). After baseline measurements, rats were randomized to exercise or no-exercise groups. A subset of animals was subjected to MRS directly after 1 h of treadmill running for measurement of total intrahepatocellular lipid (IHCL) and intramyocellular lipid (IMCL) content (n=7 lean and diabetic rats). The other animals were administered 13C-labeled lipids orally after treadmill visit (with or without exercise), followed by MRS measurements after 4 and 24 h to determine the 13C enrichment of IHCL and IMCL (n=8 per group). Total IHCL and IMCL content were 5-fold higher in diabetic versus lean rats (P<0.001). Exercise did not significantly affect IHCL content, but reduced IMCL by 25±7% and 33±4% in lean and diabetic rats (P<0.05), respectively. The uptake of dietary lipids in liver and muscle was 2.3-fold greater in diabetic versus lean rats (P<0.05). Prior exercise did not significantly modulate dietary lipid uptake into muscle, but in liver of both lean and diabetic rats, lipid uptake was 44% reduced after acute exercise (P<0.05). In conclusion, IMCL but not IHCL represents a viable substrate source during exercise in both lean and diabetic rats and exercise differentially affects dietary lipid uptake in muscle and liver.

    September 29, 2015   doi: 10.1152/ajpendo.00292.2015   open full text
  • N{varepsilon}-(carboxymethyl) lysine-induced mitochondrial fission and mitophagy causes decreased insulin secretion from beta cells.
    Lo, M.-C., Chen, M.-H., Lee, W.-S., Lu, C.-I., Chang, C.-R., Kao, S.-H., Lee, H.-M.
    AJP Endocrinology and Metabolism. September 22, 2015

    N-(carboxymethyl) lysine-conjugated bovine serum albumin (CML-BSA) is a major component of advanced glycation end-products (AGEs). We hypothesised that AGEs reduce insulin secretion from pancreatic beta cells by damaging mitochondrial functions and inducing mitophagy. Mitochondrial morphology and the occurrence of autophagy were examined in pancreatic islets of diabetic db/db mice and in cultured CML-BSA-treated insulinoma cell line RIN-m5F. In addition, effects of alpha-lipoic acid (ALA) on mitochondria in AGEs-damaged tissues were evaluated. The diabetic db/db mouse exhibited an increase in the number of autophagosomes in damaged mitochondria and receptor for AGEs (RAGE). Treatment of db/db mice with ALA for 12 weeks increased the number of mitochondria with well-organized cristae and fewer autophagosomes. Treatment of RIN-m5F cells with CML-BSA increased the level of RAGE protein and autophagosome formation, caused mitochondrial dysfunction, and decreased insulin secretion. CML-BSA also reduced mitochondrial membrane potential and ATP production, increased ROS and lipid peroxides production, and caused mitochondrial DNA deletions. Elevated fission protein dynamin-related protein 1 (Drp1) level and mitochondrial fragmentation demonstrated the unbalance of mitochondrial fusion and fission in CML-BSA-treated cells.Additionally, increased levels of Parkin and PTEN-induced putative kinase 1 (PINK1) protein suggest that fragmented mitochondria were associated with increased mitophagic activity and ALA attenuated the CML-BSA-induced mitophage formation. Our study demonstrated that CML-BSA induced mitochondrial dysfunction and mitophagy in pancreatic beta cells. The findings from this study suggest that increased concentration of AGEs may damage beta cells and reduce insulin secretion.

    September 22, 2015   doi: 10.1152/ajpendo.00151.2015   open full text
  • Cholecystokinin expression in the {beta}-cell leads to increased {beta}-cell area in aged mice and protects from streptozotocin-induced diabetes and apoptosis.
    Lavine, J. A., Kibbe, C. R., Baan, M., Sirinvaravong, S., Umhoefer, H. M., Engler, K. A., Meske, L. M., Sacotte, K. A., Erhardt, D. P., Davis, D. B.
    AJP Endocrinology and Metabolism. September 22, 2015

    Cholecystokinin (CCK) is a peptide hormone produced in the gut and brain with beneficial effects on digestion, satiety, and insulin secretion. CCK is also expressed in pancreatic β-cells, but only in models of obesity and insulin resistance. Whole-body deletion of CCK in obese mice leads to reduced β-cell mass expansion and increased apoptosis. We hypothesized that islet-derived CCK is important in protection from β-cell apoptosis. In order to determine the specific role of β-cell derived CCK in β-cell mass dynamics, we generated a transgenic mouse that expresses CCK in the β-cell in the lean state (MIP-CCK). Although this transgene contains the human growth hormone minigene, we saw no expression of human growth hormone protein in transgenic islets. We examined the ability of MIP-CCK mice to maintain β-cell mass when subjected to apoptotic stress, with advanced age and after streptozotocin treatment. Aged MIP-CCK mice have increased β-cell area. MIP-CCK mice are resistant to streptozotocin-induced diabetes and exhibit reduced β-cell apoptosis. Directed CCK overexpression in cultured β-cells also protects from cytokine-induced apoptosis. We have identified an important new paracrine/autocrine effect of CCK in protection of β-cells from apoptotic stress. Understanding the role of β-cell CCK adds to the emerging knowledge of classic gut peptides in intra-islet signaling. CCK receptor agonists are being investigated as therapeutics for obesity and diabetes. While these agonists clearly have beneficial effects on body weight and insulin sensitivity in peripheral tissues, they may also directly protect β-cells from apoptosis.

    September 22, 2015   doi: 10.1152/ajpendo.00159.2015   open full text
  • The Short Chain Fatty Acid Receptor, FFA2, contributes to gestational glucose homeostasis.
    Fuller, M., Priyadarshini, M., Gibbons, S. M., Angueira, A. R., Brodsky, M., Hayes, M. G., Kovatcheva-Datchary, P., Backhed, F., Gilbert, J. A., Lowe, W. L., Layden, B. T.
    AJP Endocrinology and Metabolism. September 22, 2015

    The structure of the human GI microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wildtype (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein coupled receptors (GPCRs) such as FFA2, and we have previously demonstrated that the Ffar2 expression is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion as compared to WT mice during, but not prior to, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Together, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.

    September 22, 2015   doi: 10.1152/ajpendo.00171.2015   open full text
  • Downregulation of Apelin in the Human Placental Chorionic Villi from Preeclamptic Pregnancies.
    Yamaleyeva, L. M., Chappell, M. C., Brosnihan, K. B., Anton, L., Caudell, D. L., Shi, S., McGee, C., Pirro, N. T., Gallagher, P. E., Taylor, R., Merrill, D. C., Mertz, H. L.
    AJP Endocrinology and Metabolism. September 22, 2015

    The role of the endogenous apelin system in pregnancy is not well understood. Apelin's actions in pregnancy are further complicated by the expression of multiple forms of the peptide. Using radioimmunoassay (RIA) alone, we established the expression of apelin content in the chorionic villi of preeclamptic (PRE) and normal pregnant women (NORM) at 36-38 weeks of gestation. Total apelin content was lower in PRE compared to NORM chorionic villi (49.7±3.4 vs.72.3±9.8 fmol/mg protein, n=20-22) and was associated with a trend for lower preproapelin mRNA in the PRE. Further characterization of apelin isoforms by HPLC-RIA was conducted in pooled samples from each group. The expression patterns of apelin peptides in NORM and PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate apelin-13 [(Pyr1)-apelin-13] was the predominant form of the peptide in NORM and PRE villi. ACE2 activity was higher in PRE villi (572.0±23.0 vs. 485.3±24.8 pmol/mg/min, n=18-22). Low dose of Ang II (1nM; 2 hours) decreased apelin release in NORM villous explants that was blocked by the AT1 receptor antagonist losartan. Moreover, losartan enhanced apelin release above the 2-hour baseline levels in both NORM and PRE villi (p<0.05). In summary, these studies are the first to demonstrate the lower apelin content in human placental chorionic villi of PRE subjects using quantitative RIA. (Pyr1)-apelin-13 is the predominant form of endogenous apelin in the chorionic villi of NORM and PRE. The potential mechanism of lower apelin expression in the PRE villi may involve a negative regulation of apelin by Ang II.

    September 22, 2015   doi: 10.1152/ajpendo.00272.2015   open full text
  • Hepatic Glucose Uptake and Disposition during Short-Term High Fat vs. High Fructose Feeding.
    Coate, K. C., Kraft, G., Moore, M. C., Smith, M. S., Ramnanan, C. J., Irimia, J. M., Roach, P. J., Farmer, B., Neal, D. W., Williams, P. E., Cherrington, A. D.
    AJP Endocrinology and Metabolism. May 27, 2014

    In dogs consuming a high-fat and -fructose diet (52% and 17% of total energy, respectively) for 4 weeks, hepatic glucose uptake (HGU) in response to hyperinsulinemia, hyperglycemia, and portal glucose delivery is markedly blunted, with reduction in glucokinase (GK) protein and glycogen synthase (GS) activity. The present study compared the impact of selective increases in dietary fat and fructose on liver glucose metabolism. Dogs consumed weight-maintaining chow (CTR) or hypercaloric high fat (HFA), or high fructose (HFR) diets diet for 4 weeks before undergoing clamp studies with infusion of somatostatin and intraportal insulin (3-4x basal) and glucagon (basal). The hepatic glucose load (HGL) was doubled during the clamp using peripheral vein (Pe) glucose infusion in the first 90 min (P1)and portal vein (4 mg•kg-1•min-1) plus Pe glucose infusion during the final 90 min (P2) During P2, HGU was 2.8±0.2, 1.0±0.2, and 0.8±0.2 mg•kg-1•min-1 in CTR, HFA, and HFR, respectively (P<0.05 for HFA and HFR vs. CTR). In comparison to CTR, hepatic GK protein and catalytic activity were reduced (P<0.05) 35% and 56%, respectively, in HFA, and 53% and 74% in HFR. Liver glycogen concentrations were 20% and 38% lower in HFA and HFR than CTR (P<0.05). Hepatic Akt phosphorylation was decreased (P<0.05) in HFA (21%) but not HFR. Thus, HFR impaired hepatic GK and glycogen more than HFA, while HFA reduced insulin signaling more than HFR. HFA and HFR effects were not additive, suggesting they act via the same mechanism or their effects converge at a saturable step.

    May 27, 2014   doi: 10.1152/ajpendo.00083.2014   open full text
  • Impairments of hepatic gluconeogenesis and ketogenesis in PPAR{alpha}-deficient neonatal mice.
    Cotter, D. G., Ercal, B., d'Avignon, D. A., Dietzen, D. J., Crawford, P. A.
    AJP Endocrinology and Metabolism. May 27, 2014

    Peroxisome proliferator activated receptor alpha (PPARα) is a master transcriptional regulator of hepatic metabolism, and mediates the adaptive response to fasting. Here we demonstrate the roles for PPARα in hepatic metabolic adaptations to birth. Like fasting, nutrient supply is abruptly altered at birth when a transplacental source of carbohydrates is replaced by a high-fat, low-carbohydrate milk diet. PPARα-knockout (KO) neonatal mice exhibit relative hypoglycemia due to impaired conversion of glycerol to glucose. While hepatic expression of fatty acyl-CoA dehydrogenases is imparied in PPARα neonates, these animals exhibit normal blood acylcarnitine profiles. Furthermore, quantitative metabolic fate mapping of the medium-chain fatty acid [13C]octanoate in neonatal mouse livers revealed normal contribution of this fatty acid to the hepatic TCA cycle. Interestingly, octanoate-derived carbon labeled glucose uniquely in livers of PPARα-KO neonates. Relative hypoketonemia in newborn PPARα-KO animals could be mechanistically linked to a 50% decrease in de novo hepatic ketogenesis from labeled octanoate. Decreased ketogenesis was associated with diminished mRNA and protein abundance of the fate-committing ketogenic enzyme mitochondrial 3-hydroxymethylglutaryl-CoA synthase (HMGCS2) and decreased protein abundance of the ketogenic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1). Finally, hepatic triglyceride and free fatty acid concentrations were increased 6.9- and 2.7-fold, respectively in suckling PPARα-KO neonates. Together, these findings indicate a primary defect of gluconeogenesis from glycerol, and an important role for PPARα-dependent ketogenesis in the disposal of hepatic fatty acids during the neonatal period.

    May 27, 2014   doi: 10.1152/ajpendo.00087.2014   open full text
  • Aetiology of the membrane potential of rat white fat adipocytes.
    Bentley, D. C., Pulbutr, P., Chan, S., Smith, P. A.
    AJP Endocrinology and Metabolism. May 27, 2014

    The plasmamembrane potential (Vm) is key to many physiological processes, however its ionic aetiology in white fat adipocytes is poorly characterised. To address this question, we have employed the perforated patch and cell-attached patch-clamp methods in isolated primary white fat adipocytes and their cellular model: 3T3-L1. The resting Vm of primary and 3T3-L1 adipocytes were -32.1±1.2mV (n=95) and -28.8±1.2mV (n=87), respectively. Vm was independent of cell size and fat content. Elevation of extracellular [K+] to 50mM by equimolar substitution of bath Na+ did not affect Vm, whereas substitution of bath Na+ with the membrane impermeant cation N-methyl-D-glucamine+ hyperpolarized Vm by 16mV, data indicative of a non-selective cation permeability. Substitution of 133mM extracellular Cl- with gluconate depolarised Vm by 25mV, whereas Cl- substitution with I- caused a -9mV hyperpolarization. Isoprenaline (10µM) but not insulin (100nM) significantly depolarized Vm. Single-channel ion activity was voltage independent; currents were indicative for Cl- with an inward slope conductance of 16±1.3pS (n=11) and a reversal potential close to the Cl- equilibrium potential: -29±1.6mV. Reduction of extracellular Cl- elevated the intracellular Ca2+. In conclusion, the Vm of white fat adipocyte is well described by the Goldman-Hodgkin-Katz equation with a predominant permeability to Cl-, where its biophysical and single-channel properties suggest a volume-sensitive anion channel identity. Consequently, changes in serum Cl- homeostasis or the adipocyte's permeability to this anion via drugs will affect its Vm, intracellular Ca2+ and ultimately its function and role in metabolic control.

    May 27, 2014   doi: 10.1152/ajpendo.00446.2013   open full text
  • Linoleic and {alpha}-linolenic acid both prevent insulin resistance but have divergent impacts on skeletal muscle mitochondrial bioenergetics in obese Zucker rats.
    Matravadia, S., Herbst, E. A., Jain, S. S., Mutch, D. M., Holloway, G. P.
    AJP Endocrinology and Metabolism. May 20, 2014

    The therapeutic use of polyunsaturated fatty acids (PUFA) in preserving insulin sensitivity has gained interest in recent decades; however, the roles of linoleic acid (LA) and α-linolenic acid (ALA) remain poorly understood. We investigated the efficacy of diets enriched with either LA or ALA on attenuating the development of insulin resistance (IR) in obesity. Following a twelve-week intervention, LA and ALA both prevented the shift towards an IR phenotype and maintained muscle-specific insulin sensitivity otherwise lost in obese control animals. The beneficial effects of ALA were independent of changes in skeletal muscle mitochondrial content and oxidative capacity, as obese control and ALA treated rats showed similar increases in these parameters. However, ALA increased the propensity for mitochondrial H2O2 emission and catalase content within whole-muscle, and reduced markers of oxidative stress (4-HNE and carbonyl content). In contrast, LA prevented changes in markers of mitochondrial content, respiratory function, H2O2 emission and oxidative stress in obese animals, thereby resembling levels seen in lean animals. Together, our data suggests that LA and ALA are efficacious in preventing IR but have divergent impacts on skeletal muscle mitochondrial content and function. Moreover, we propose that LA has value in preserving insulin sensitivity in the development of obesity; thereby challenging the classical view that n-6 PUFAs are detrimental.

    May 20, 2014   doi: 10.1152/ajpendo.00032.2014   open full text
  • Embryo number and periconceptional undernutrition in the sheep have differential effects on adrenal epigenotype, growth and development.
    Williams-Wyss, O., Zhang, S., MacLaughlin, S. M., Kleemann, D. O., Walker, S. K., Suter, C. M., Cropley, J. E., Morrison, J. L., Roberts, C. T., McMillen, I. C.
    AJP Endocrinology and Metabolism. May 20, 2014

    Exposure to poor maternal nutrition around the time of conception results in an early prepartum activation of the fetal pituitary-adrenal axis and in increased adrenal growth and stress response after birth associated with epigenetic changes in a differentially methylated region (DMR) of adrenal IGF2/H19. We have determined the effects of maternal undernutrition during the periconceptional period (PCUN: 70% of control intake from 60 days before until 6 days after conception) and early preimplantation period (PIUN: 70% of control intake for 6 days after conception on fetal plasma ACTH and cortisol concentrations and fetal adrenal ACTHR, StAR, 3βHSD, CYP11B, CYP17, TGFβ1, IGF1, IGF1R, IGF2, and IGF2R mRNA expression and the methylation level of sites within the DMRs of IGF2/H19 and IGF2R in the adrenal of twin and singleton fetuses at 136-138 d gestation. Being a twin resulted in a delayed prepartum increase in fetal ACTH and in a lower cortisol response to CRH in the control, but not PCUN and PIUN groups. PCUN, but not PIUN, resulted in an increase in adrenal weight and CYP17 expression in singletons, a decrease in adrenal IGF2 expression in singletons and an increase in adrenal IGF2R expression in both twins and singletons. IGF2/H19 and IGF2R DMR methylation levels and ACTHR expression were lower in the twinadrenal. Thus exposure of the oocyte and embryo to maternal undernutrition or to the environment of a twin pregnancy have differential effects on epigenetic and other factors which regulate fetal adrenal growth and IGF2 and IGF2R expression.

    May 20, 2014   doi: 10.1152/ajpendo.00051.2012   open full text
  • Consumption of a Western-style diet during pregnancy impairs offspring islet vascularization in a Japanese Macaque model.
    Pound, L. D., Comstock, S. M., Grove, K. L.
    AJP Endocrinology and Metabolism. May 20, 2014

    Children exposed to a maternal Western-style diet in utero have an increased risk of developing type 2 diabetes. Understanding the mechanisms and an investigation of possible interventions are critical to reversing this phenomenon. We examined the impact of maternal Western-style diet consumption on the development of islet vascularization and innervation, both critical to normal islet function, in fetal and juvenile offspring. Furthermore, we assessed whether improved dietary intake or resveratrol supplementation could ameliorate the harmful consequences of Western-style diet consumption during pregnancy. Adult female Japanese Macaques were maintained on a control or Western-style diet for 4-7 years. One cohort of dams was switched back onto a control diet while another cohort received resveratrol supplementation throughout gestation. Pregnancies were terminated in the early third trimester by C-section or offspring were born naturally and sent to necropsy at 1 year of age. Western-style diet consumption resulted in impaired fetal islet capillary density and sympathetic islet innervation. Furthermore, this reduction in vascularization persisted in the juvenile offspring. This effect is independent of changes in the expression of key angiogenic markers. Diet reversal normalized islet vascularization to control offspring levels while resveratrol supplementation caused a significant increase in capillary density above controls. These data provide a novel mechanism by which maternal Western-style diet consumption leads to increased susceptibility to type 2 diabetes in the offspring. Importantly, an improved maternal diet may mitigate these harmful effects. However, until the long-term consequences of increased vascularization can be determined, resveratrol use during pregnancy is not advised.

    May 20, 2014   doi: 10.1152/ajpendo.00131.2014   open full text
  • Training Status Diverges Muscle Diacylglycerol Accumulation During Free Fatty Acid Elevation.
    Chow, L. S., Mashek, D. G., Austin, E., Eberly, L. E., Persson, X.-M., Mashek, M. T., Seaquist, E. R., Jensen, M. D.
    AJP Endocrinology and Metabolism. May 20, 2014

    Aims: How endurance training alters muscle lipid metabolism while preserving insulin sensitivity remains unclear. Because acute free fatty acid (FFA) elevation by lipid infusion reduces insulin sensitivity, we hypothesized that training status would alter accumulation of muscle triacylglycerol (TAG), diacylglycerol (DAG), ceramide, and acylcarnitine during acute FFA elevation. Methods: Trained (n=15) and sedentary (n=13) participants matched for age, sex, and BMI received either a 6-hour infusion of lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) during a hyperinsulinemic-euglycemic clamp. Muscle biopsies were taken at 0, 120, and 360 min after infusion initiation to measure intramyocellular concentrations of TAG, DAG, ceramides, and acylcarnitines by liquid chromatography/tandem mass spectrometry. Results: Trained participants had a higher VO2 max and insulin sensitivity than sedentary participants. The lipid infusion produced comparable elevation of FFA [594 ± 90 (SE) µmol/L in trained, 721 ± 30 (SE) µmol/L in sedentary, p = 0.4] and decline in insulin sensitivity (-44.7% trained vs. -47.2% sedentary, p=0.89). In both groups, lipid infusion increased the linoleic and linolenic acid content of TAG without changing total TAG. In the sedentary group, lipid infusion increased total, oleic, linoleic acid and linolenic acid content of DAG. Regardless of training status, lipid infusion did not alter total ceramide, saturated ceramide, palmitoyl-carnitine or oleoyl-carnitine. Conclusion: During acute FFA elevation, trained adults have a similar decline in insulin sensitivity with less accumulation of muscle DAG than sedentary adults, suggesting that lipid-induced insulin resistance can occur without elevation of total muscle DAG.

    May 20, 2014   doi: 10.1152/ajpendo.00166.2014   open full text
  • Reactive oxygen species induce the association of SHP-1 with c-Src and the oxidation of both to enhance osteoclast survival.
    Ke, K., Sul, O.-J., Choi, E.-K., Safdar, A. M., Kim, E.-S., Choi, H.-S.
    AJP Endocrinology and Metabolism. May 13, 2014

    Loss of ovarian function causes oxidative stress as well as bone loss. We hypothesized that reactive oxygen species (ROS) induced by the failure of ovarian function are responsible for the bone loss by increasing the number of osteoclasts (OC). We found that ROS enhanced OC survival via Src homology 2 domain-containing phosphatase-1 (SHP-1), c-Src, Akt, and ERK. ROS induced the association of SHP-1 with c-Src, as well as the oxidation of c-Src and SHP-1. This resulted in inactivation of SHP-1and activation of c-Src via phosphorylation of Tyr 416. Knock-down of c-Src or SHP-1 abolished the effect of ROS on OC survival. Moreover, down-regulation of SHP-1 up-regulated activation of c-Src, Akt, and ERK in the absence of any stimulus, suggesting that inactivation of SHP-1 is required for OC survival. We demonstrated that the association and oxidation of c-Src and SHP-1 by ROS are key steps in enhancing OC survival, which are responsible for increased bone loss when ovarian function ceases.

    May 13, 2014   doi: 10.1152/ajpendo.00044.2014   open full text
  • Citrulline does not enhance blood flow, microvascular circulation, or myofibrillar protein synthesis in elderly men at rest or following exercise.
    Churchward-Venne, T. A., Cotie, L. M., MacDonald, M. J., Mitchell, C. J., Prior, T., Baker, S. K., Phillips, S. M.
    AJP Endocrinology and Metabolism. May 13, 2014

    Ageing is associated with anabolic resistance; a reduced sensitivity of myofibrillar protein synthesis (MPS) to postprandial hyperaminoacidemia, particularly with low protein doses. Impairments in postprandial skeletal muscle blood flow and/or microvascular perfusion with hyperaminoacidemia and hyperinsulinemia may contribute to anabolic resistance. We examined whether providing citrulline, a precursor for arginine and nitric oxide synthesis, would increase arterial blood flow, skeletal muscle microvascular perfusion, MPS, and signalling through mTORC1. Twenty one elderly males (65-80 y) completed acute unilateral resistance exercise prior to being assigned to ingest a: high dose (45g) of whey protein (WHEY), or a low dose (15g) of whey protein with 10g of citrulline (WHEY+CIT), or with 10g of non-essential amino acids (WHEY+NEAA). A primed continuous infusion of L-[ring-13C6] phenylalanine with serial muscle biopsies was used to measure MPS and protein phosphorylation, while ultrasound was used to measure microvascular circulation under basal and postprandial conditions in both a rested (FED) and exercised (EX-FED) leg. Argininemia was greater in WHEY+CIT vs. WHEY and WHEY+NEAA from 30-300 min post-exercise (P<0.001), but there were no treatment differences in blood flow, or microvascular perfusion (all P>0.05). Phosphorylation of p70S6kThr389 was greater in WHEY vs. WHEY+NEAA (P=0.02). Postprandial MPS was greater in WHEY vs. WHEY+CIT and WHEY+NEAA under both FED (WHEY: ~128%; WHEY+CIT: ~56%; WHEY+NEAA: ~38%) and EX-FED (WHEY: ~251%; WHEY+CIT: ~124%; WHEY+NEAA: ~108%) conditions (P=0.003). Citrulline co-ingestion with a low quantity of protein was ineffective in augmenting the anabolic properties of protein compared to non-essential amino acids.

    May 13, 2014   doi: 10.1152/ajpendo.00096.2014   open full text
  • Mediobasal hypothalamic PTEN modulates hepatic insulin resistance independently of food intake in rats.
    Sumita, T., Ono, H., Suzuki, T., Sakai, G., Inukai, K., Katagiri, H., Asano, T., Katayama, S., Awata, T.
    AJP Endocrinology and Metabolism. May 13, 2014

    PTEN (phosphatase and tensin homolog) dephosphorylates phosphatidylinositol 3,4,5-triphosphate and antagonizes PI 3-kinase. Insulin acts in the mediobasal hypothalamus (MBH) not only to suppress food intake and weight gain, but also to improve glucose metabolism, via PI 3-kinase activation. Thus, blocking hypothalamic PTEN is a potential target for treating obesity as well as diabetes. However, genetic modification of PTEN in specific neuronal populations in the MBH yielded complex results, and no postnatal intervention for hypothalamic PTEN has yet been reported. In order to elucidate how postnatal modification of hypothalamic PTEN influences food intake as well as glucose metabolism, we bidirectionally altered PTEN activity in the MBH of rats by adenoviral gene delivery. Inhibition of MBH PTEN activity reduced food intake and weight gain, while constitutive activation of PTEN tended to induce the opposite effects. Interestingly, the effects of MBH PTEN intervention on food intake and body weight were blunted by high-fat feeding. However, MBH PTEN blockade improved hepatic insulin sensitivity even under high-fat fed conditions. On the other hand, constitutive activation of MBH PTEN induced hepatic insulin resistance. Hepatic Akt phosphorylation and the G6Pase expression level were bidirectionally modulated by MBH PTEN intervention. These results demonstrate that PTEN in the MBH regulates hepatic insulin sensitivity, independently of the effects on food intake and weight gain. Therefore, hypothalamic PTEN is a promising target for treating insulin resistance even in states of over-nutrition.

    May 13, 2014   doi: 10.1152/ajpendo.00361.2013   open full text
  • FFA-induced Hepatic Insulin Resistance in vivo is mediated by PKC-{delta}, NADPH Oxidase, and Oxidative Stress.
    Pereira, S., Park, E., Mori, Y., Haber, C. A., Han, P., Uchida, T., Stavar, L., Oprescu, A. I., Koulajian, K., Ivovic, A., Yu, Z., Li, D., Bowman, T. A., Dewald, J., El-Benna, J., Brindley, D. N., Gutierrez-Juarez, R., Lam, T. K. T., Najjar, S. M., McKay, R. A., Bhanot, S., Fantus, I. G., Giacca, A.
    AJP Endocrinology and Metabolism. May 13, 2014

    Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKC- and oxidative stress play causal roles in this process and whether this occurs via IKKβ- and JNK-dependent pathways. Rats received 7h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2h of the infusion, hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IBα content, increased JNK phosphorylation (markers of IKKβ and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver, without affecting IH-induced hepatic PKC- activation. Furthermore, antisense oligonucleotide against PKC- prevented IH-induced phosphorylation of p47phox (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similar to NAC. These results demonstrate that PKC-, NADPH oxidase and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA -> PKC- -> NADPH oxidase and oxidative stress -> IKKβ/JNK -> impaired hepatic insulin signaling.

    May 13, 2014   doi: 10.1152/ajpendo.00436.2013   open full text
  • miR-24 Regulates Menin in the Endocrine Pancreas.
    Vijayaraghavan, J., Maggi, E. C., Crabtree, J. S.
    AJP Endocrinology and Metabolism. May 13, 2014

    Menin, the product of the MEN1 gene, functions as a tumor suppressor and was first identified in 1997 due to its causative role in the endocrine tumor disorder Multiple Endocrine Neoplasia, type 1 (MEN1). More recently, menin has been identified as a key player in pancreatic islet biology with the observation of an inverse relationship between menin levels and pancreatic islet proliferation. However, the factors regulating menin and the MEN1 gene in the pancreas are poorly understood. Here we describe the regulation of menin by miR-24 and demonstrate that miR-24 directly decreases menin levels and impacts downstream cell cycle inhibitors in MIN6 insulinoma cells and in βlox5 immortalized beta cells. This regulation of menin impacts cell viability and proliferation in βlox5 cells. Furthermore, our data show a feedback regulation between miR-24 and menin that is present in the pancreas suggesting that miR-24 regulates menin levels in the pancreatic islet.

    May 13, 2014   doi: 10.1152/ajpendo.00542.2013   open full text
  • Targeted metabolomics and mathematical modeling demonstrate that vitamin B-6 restriction alters one-carbon metabolism in cultured HepG2 cells.
    da Silva, V. R., Ralat, M. A., Quinlivan, E. P., DeRatt, B. N., Garrett, T. J., Chi, Y.-Y., Nijhout, H. F., Reed, M. C., Gregory, J. F.
    AJP Endocrinology and Metabolism. May 13, 2014

    Low vitamin B-6 nutritional status is associated with increased risk for cardiovascular disease and certain cancers. Pyridoxal 5'-phosphate (PLP) serves as a coenzyme in many cellular processes including several reactions in one-carbon (1C) metabolism and the transsulfuration pathway of homocysteine catabolism. To assess the effect of vitamin B-6 deficiency on these processes and associated pathways, we conducted quantitative analysis of 1C metabolites including tetrahydrofolate species in HepG2 cells cultured in various concentrations of pyridoxal. These results were compared with predictions of a mathematical model of 1C metabolism simulating effects of vitamin B-6 deficiency. In cells cultured in vitamin B6 deficient media (25 or 35 nmol/L pyridoxal), we observed >200% higher concentrations of betaine P<0.05) and creatinine (P<0.05) and >60% lower concentrations of creatine (P<0.05) and 5,10-methenyltetrahydrofolate (P<0.05) compared to cells cultured in media containing intermediate (65 nmol/L) or the supraphysiological 2015 nmol/L pyridoxal. Cystathionine, cysteine, glutathione, and cysteinylglycine, which are components of the transsulfuration pathway and subsequent reactions, exhibited greater concentrations at the two lower vitamin B-6 concentrations. Partial least squares-discriminant analysis showed differences in overall profiles between cells cultured in 25 and 35 nmol/L pyridoxal versus those in 65 and 2015 nmol/L pyridoxal. Mathematical model predictions aligned with analytically derived results. These data reveal pronounced effects of vitamin B-6 deficiency on 1C-related metabolites including previously unexpected secondary effects on creatine. These results compliment metabolomic studies in humans demonstrating extended metabolic effects of vitamin B-6 insufficiency.

    May 13, 2014   doi: 10.1152/ajpendo.00697.2013   open full text
  • Activation of glucagon-like peptide-1 receptor inhibits growth and promotes apoptosis of human pancreatic cancer cells in a cAMP-dependent manner.
    Zhao, H., Wei, R., Wang, L., Tian, Q., Tao, M., Ke, J., Liu, Y., Hou, W., Zhang, L., Yang, J., Hong, T.
    AJP Endocrinology and Metabolism. May 06, 2014

    Glucagon-like peptide-1 (GLP-1) promotes pancreatic β-cell regeneration through GLP-1 receptor (GLP-1R) activation. However, whether it promotes exocrine pancreas growth and thereby increases risk of pancreatic cancer has been a topic of debate in recent years. Clinical data and animal studies published so far have been controversial. In the present study, we report that GLP-1R activation with liraglutide inhibited growth and promoted apoptosis in human pancreatic cancer cell lines in vitro and attenuated pancreatic tumor growth in a mouse xenograft model in vivo. These effects of liraglutide were mediated through activation of cAMP production and consequent inhibition of Akt and Erk1/2 signaling pathways in a GLP-1R-dependent manner. Moreover, we examined GLP-1R expression in human pancreatic cancer tissues, and found that 43.3% of tumor tissues were GLP-1R-null. In the GLP-1R-positive tumor tissues (56.7%), the level of GLP-1R was lower compared with that in tumor-adjacent normal pancreatic tissues. Furthermore, the GLP-1R-positive tumors were significantly smaller than the GLP-1R-null tumors. Our study shows for the first time that GLP-1R activation has a cytoreductive effect on human pancreatic cancer cells in vitro and in vivo, which may help address safety concerns of GLP-1-based therapies in the context of human pancreatic cancer.

    May 06, 2014   doi: 10.1152/ajpendo.00017.2014   open full text
  • mTORC1 and JNK Coordinate Phosphorylation of the p70S6K1 Autoinhibitory Domain in Skeletal Muscle Following Functional Overloading.
    Martin, T. D., Dennis, M. D., Gordon, B. S., Kimball, S. R., Jefferson, L. S.
    AJP Endocrinology and Metabolism. May 06, 2014

    The present project was designed to investigate phosphorylation of p70S6K1 in an animal model of skeletal muscle overload. Within 24 h of male Sprague Dawley rats undergoing unilateral tenotomy to induce functional overloading of the plantaris muscle, phosphorylation of the Thr389 and Thr421/Ser424 sites on p70S6K1 was significantly elevated. Since the Thr421/Ser424 sites are purportedly mTORC1-independent, we sought to identify the kinase(s) responsible for their phosphorylation. Initially, we used IGF1 treatment of serum-deprived HEK293E cells as an in vitro model system, because IGF1 promotes phosphorylation of p70S6K1 on both the Thr389 and Thr421/Ser424 sites in skeletal muscle and in cells in culture. We found that, whereas the mTOR inhibitor TORIN2 prevented the IGF1-induced phosphorylation of the Thr421/Ser424 sites, it surprisingly enhanced phosphorylation of these sites during serum deprivation. JNK inhibition with SP600125 attenuated phosphorylation of the Thr421/Ser424 sites, and in combination with TORIN2 both the effect of IGF1 and the enhanced Thr421/Ser424 phosphorylation during serum deprivation were ablated. In contrast, both JNK activation with anisomycin and knockdown of the mTORC2 subunit rictor specifically stimulated phosphorylation of the Thr421/Ser424 sites, suggesting that mTORC2 represses JNK-mediated phosphorylation of these sites. The role of JNK in mediating p70S6K1 phosphorylation was confirmed in the animal model noted above, where rats treated with SP600125 exhibited attenuated Thr421/Ser424 phosphorylation. Overall, the results provide evidence that the mTORC1 and JNK signaling pathways coordinate the site-specific phosphorylation of p70S6K1. They also identify a novel role for mTORC1 and mTORC2 in the inhibition of JNK.

    May 06, 2014   doi: 10.1152/ajpendo.00064.2014   open full text
  • Mitochondria: A Possible Nexus for the Regulation of Energy Homeostasis by the Endocannabinoid System?
    Lipina, C., Irving, A. J., Hundal, H. S.
    AJP Endocrinology and Metabolism. May 06, 2014

    The endocannabinoid system (ECS) regulates numerous cellular and physiological processes through the activation of receptors targeted by endogenously produced ligands called endocannabinoids. Importantly, this signalling system is known to play an important role in modulating energy balance and glucose homeostasis. For example, current evidence indicates that the ECS becomes overactive during obesity whereby its central and peripheral stimulation drives metabolic processes that mimic the metabolic syndrome. Herein, we examine the role of the ECS in modulating the function of mitochondria which play a pivotal role in maintaining cellular and systemic energy homeostasis, in large part due to their ability to tightly coordinate glucose and lipid utilisation. Because of this, mitochondrial dysfunction is often associated with peripheral insulin resistance and glucose intolerance, as well as the manifestation of excess lipid accumulation in the obese state. This review aims to highlight the different ways through which the ECS may impact upon mitochondrial abundance and/or oxidative capacity, and where possible, relate these findings to obesity-induced perturbations in metabolic function. Furthermore, we explore the potential implications of these findings in terms of the pathogenesis of metabolic disorders and how these may be used to strategically develop therapies targeting the ECS.

    May 06, 2014   doi: 10.1152/ajpendo.00100.2014   open full text
  • A Novel Oral Dual Amylin and Calcitonin Receptor Agonist (KBP-042) Exerts Anti-Obesity and Anti-Diabetic Effects in Rats.
    Andreassen, K. V., Feigh, M., Hjuler, S. T., Gydesen, S., Henriksen, J. E., Beck-Nielsen, H., Christiansen, C., Karsdal, M. A., Henriksen, K.
    AJP Endocrinology and Metabolism. May 06, 2014

    The present study investigated a novel oral dual amylin and calcitonin receptor agonist (DACRA), KBP-042, in head-to-head comparison with salmon calcitonin (sCT) in regards to in vitro receptor pharmacology, ex vivo pancreatic islet studies and in vivo proof of concept studies in diet-induced obese (DIO) and Zucker diabetic fatty (ZDF) rats. In vitro, KBP-042 demonstrated superior binding affinity and activation of amylin and calcitonin receptors, and ex vivo, KBP-042 exerted inhibitory action on stimulated insulin and glucagon release from isolated islets. In vivo, KBP-042 induced a superior and pronounced reduction in food intake in conjunction with a sustained pair-fed corrected weight-loss in DIO rats. Concomitantly, KBP-042 improved glucose homeostasis and reduced hyperinsulinemia and hyperleptinemia in conjunction with enhanced insulin sensitivity. In ZDF rats, KBP-042 induced a superior attenuation of diabetic hyperglycemia and alleviated impaired glucose and insulin tolerance. Concomitantly, KBP-042 preserved insulinotropic and induced glucagonostatic action, ultimately preserving pancreatic insulin and glucagon content. In conclusion, oral KBP-042 is a novel DACRA, which exerts anti-obesity and anti-diabetic efficacy by dual modulation of insulin sensitivity and directly decelerating stress on the pancreatic alpha and beta-cells. These results could provide the basis for oral KBP-042 as a novel therapeutic agent in type 2 diabetes.

    May 06, 2014   doi: 10.1152/ajpendo.00121.2014   open full text
  • Insulin Sensitivity Is Inversely Related To Cellular Energy Status, As Revealed By Biotin Deprivation.
    Salvador-Adriano, A., Vargas-Chavez, S., Hernandez-Vazquez, A. d. J., Ortega-Cuellar, D., Tovar, A. R., Velazquez-Arellano, A.
    AJP Endocrinology and Metabolism. May 06, 2014

    We have reported an early decrease of glycemia in rats fed a biotin-deficient diet, with reduced cellular ATP levels and suggesting increased insulin sensitivity. Here we show that biotin deprived rats are more tolerant to glucose as shown by both oral and intraperitoneal glucose tolerance tests, during which insulin plasma levels were significantly diminished in deficient rats compared to controls. Biotin-deficient rats had lower blood glucose concentrations during intraperitoneal insulin sensitivity tests than controls. Furthermore, more glucose was infused to maintain euglycemia in the biotin deficient rats during hyperinsulinemic euglycemic clamps studies. These results demonstrate augmented sensitivity to insulin in biotin-deprived rats. They are most likely consequence of an insulin-independent effect of AMPK activation on GLUT4 membrane translocation with increased glucose uptake. In biotin deficient cultured L6 muscle cells, there was increased phosphorylation of the energy sensor AMPK. We have now confirmed the augmented AMPK activation in both biotin-deprived in vivo muscle and in cultured muscle cells. In these cells, glucose uptake is increased by AMPK activation by AICAR, and is diminished by its knockdown by the specific siRNAs directed against its a1 and a2 catalytic subunits; all of these effects being largely independent of the activity of the insulin signaling pathway that was inhibited with Wortmannin. The enhanced insulin sensitivity in biotin deficiency likely has adaptive value for organisms due to the hormone promotion of uptake and utilization not only of glucose, but other nutrients such as branched-chain amino acids, whose deficiency has been reported to increase insulin tolerance.

    May 06, 2014   doi: 10.1152/ajpendo.00442.2013   open full text
  • Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization.
    Xie, X., Langlais, P., Zhang, X., Heckmann, B. L., Saarinen, A. M., Mandarino, L. J., Liu, J.
    AJP Endocrinology and Metabolism. May 06, 2014

    Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, is long known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr-372 resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. While it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr-372 eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr-372 to Ala gave a protein that bound LDs and functioned the same as the wild type protein. In non-stimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL while the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accordance, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr-372 as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL.

    May 06, 2014   doi: 10.1152/ajpendo.00663.2013   open full text
  • Sulforaphane reduction of testicular apoptotic cell death in diabetic mice is associated with the up-regulation of Nrf2 expression and function.
    Wang, Y., Zhang, Z., Guo, W., Sun, W., Miao, X., Wu, H., Cong, X., Wintergerst, K. A., Kong, X., Cai, L.
    AJP Endocrinology and Metabolism. May 06, 2014

    Diabetes-induced testicular cell death is predominantly due to oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an important transcription factor in controlling the anti-oxidative system and is inducible by sulforaphane (SFN). To test whether SFN prevents diabetes-induced testicular cell death, an insulin-defective stage of type 2 diabetes (IDS-T2DM) was induced in mice. This was accomplished by feeding them a high-fat diet (HFD) for 3 months to induce insulin resistance, and then giving one intraperitoneal injection of streptozotocin to induce hyperglycemia while age-matched control mice were fed a normal diet (ND). IDS-T2DM and ND-fed control mice were then further subdivided into those with or without 4-month SFN treatment. IDS-T2DM induced significant increases in testicular cell death presumably through receptor and mitochondrial pathways, shown by increased ratio of Bax/Bcl2 expression and cleavage of caspase-3 and caspase-8 without significant change of endoplasmic reticulum stress. Diabetes also significantly increased testicular oxidative damage and inflammation. All these diabetic effects were significantly prevented by SFN treatment with up-regulated Nrf2 expression. These results suggest that IDS-T2DM induces testicular cell death presumably through caspase-8 activation and mitochondria-mediated cell death pathways, and also by significantly down-regulating testicular Nrf2 expression and function. SFN up-regulates testicular Nrf2 expression, and its target antioxidant expression, which was associated with significant protection of the testis from IDS-T2DM-induced germ cell death.

    May 06, 2014   doi: 10.1152/ajpendo.00702.2013   open full text
  • Central Adiponectin Administration Reveals New Regulatory Mechanisms of Bone Metabolism in Mice.
    Wu, Y., Tu, Q., Valverde, P., Zhang, J., Murray, D., Dong, L. Q., Cheng, J., Jiang, H., Rios, M., Morgan, E., Tang, Z., Chen, J.
    AJP Endocrinology and Metabolism. April 29, 2014

    Adiponectin (APN), the most abundant adipocyte-secreted adipokine regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but still not clearly understood. In the present study, we found APN knockout (APN-KO) mice fed on a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared to wild-type (WT) mice. APN intracerebroventricular (ICV) infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), epinephrine and norepinephrine serum levels, and osteoclast numbers, while increasing osteoblast osteogenic markers expression and trabecular bone mass in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART) and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions, but decreased hypothalamic cannabinoid receptor (CB)-1 expression. Treatment of immortalized mouse neurons with APN, demonstrated that APN-mediated effects on TPH2, CART and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation and promote osteoblastic commitment of BMSC.

    April 29, 2014   doi: 10.1152/ajpendo.00048.2014   open full text
  • Running exercise alleviates trabecular bone loss and osteopenia in hemizygous {beta}-globin knockout thalassemic mice.
    Thongchote, K., Svasti, S., Teerapornpuntakit, J., Krishnamra, N., Charoenphandhu, N.
    AJP Endocrinology and Metabolism. April 29, 2014

    A marked decrease in β-globin production led to β-thalassemia, a hereditary anemic disease associated with bone marrow expansion, bone erosion, and osteoporosis. Herein, we aimed to investigate changes in bone mineral density (BMD) and trabecular microstructure in hemizygous β-globin knockout thalassemic (BKO) mice, and to determine whether endurance running (60 min/day, 5 days/week for 12 weeks in running wheels) could effectively alleviate bone loss in BKO mice. Both male and female BKO mice (1-2 months old) showed growth retardation as indicated by smaller body weight and femoral length compared to their wild-type littermates. A decrease in BMD was more severe in female than male BKO mice. Bone histomorphometry revealed that BKO mice had decreases in trabecular bone volume, trabecular number, and trabecular thickness, presumably due to suppression of osteoblast-mediated bone formation and activation of osteoclast-mediated bone resorption, the latter of which was consistent with elevated serum levels of osteoclastogenic cytokines, interleukin-1α and -1β. As determined by peripheral quantitative computed tomography, running increased cortical density and thickness in the femoral and tibial diaphyses of BKO mice when compared to those of sedentary BKO mice. Several histomorphometric parameters suggested an enhancement of bone formation (e.g., increased mineral apposition rate) and suppression of bone resorption (e.g., decreased osteoclast surface), which led to increases in trabecular bone volume and trabecular thickness in running BKO mice. In conclusion, BKO mice exhibited pervasive osteopenia and impaired bone microstructure, while running exercise appeared to be an effective intervention in alleviating bone microstructural defect in β-thalassemia.

    April 29, 2014   doi: 10.1152/ajpendo.00111.2014   open full text
  • Augmented expression and secretion of adipose derived PEDF does not alter local angiogenesis or contribute to the development of systemic metabolic derangements.
    Lakeland, T. V., Borg, M. L., Matzaris, M., Abdelkader, A., Evans, R. G., Watt, M. J.
    AJP Endocrinology and Metabolism. April 22, 2014

    Impaired coupling of adipose tissue expansion and vascularization is proposed to lead to adipocyte hypoxia and inflammation, which in turn contributes to systemic metabolic derangements. Pigment epithelium-derived factor (PEDF) is a powerful anti-angiogenic factor that is secreted by adipocytes, is elevated in obesity and is implicated in the development of insulin resistance. We explored the angiogenic and metabolic role of adipose-derived PEDF through in vivo studies of mice with overexpression of PEDF in adipocytes (PEDF-aP2). PEDF expression in white adipocytes and PEDF secretion from adipose tissue was increased in transgenic mice, but circulating levels of PEDF were not increased. Overexpression of PEDF did not alter vascularization, the partial pressure of O2, cellular hypoxia or gene expression of inflammatory markers in adipose tissue. Energy expenditure and metabolic substrate utilization, body mass and adiposity were not altered in PEDF-aP2 mice. Whole body glycemic control was normal as assessed by glucose and insulin tolerance tests and adipocyte-specific glucose uptake was unaffected by PEDF overexpression. Adipocyte lipolysis was increased in PEDF-aP2 mice and was associated with increased adipose triglyceride lipase and decreased perilipin 1 expression. Experiments conducted in mice rendered obese by high-fat feeding showed no differences between PEDF-aP2 and wildtype mice for body mass, adiposity, whole body energy expenditure, glucose tolerance, or adipose tissue oxygenation. Together, these data indicate that adipocyte-generated PEDF enhances lipolysis but question the role of PEDF as a major anti-angiogenic or pro-inflammatory mediator in adipose tissue in vivo.

    April 22, 2014   doi: 10.1152/ajpendo.00046.2014   open full text
  • Mice Lacking NOX2 are Hyperphagic and Store Fat Preferentially in the Liver.
    Costford, S. R., Castro-Alves, J., Chan, K. L., Bailey, L. J., Woo, M., Belsham, D. D., Brumell, J. H., Klip, A.
    AJP Endocrinology and Metabolism. April 22, 2014

    Chronic low-grade inflammation is an important contributor to the development of insulin resistance, a hallmark of type 2 diabetes mellitus (T2DM). Obesity and high fat feeding lead to infiltration of immune cells into metabolic tissues, promoting inflammation and insulin resistance. We hypothesized that macrophages from mice lacking NOX2 (Cybb), an essential component of the NADPH oxidase complex highly expressed in macrophages and associated with their inflammatory response, would be less inflammatory and that these mice would be protected from the development of high fat-induced insulin resistance. Bone marrow-derived macrophages from NOX2-knockout (NOX2-KO) mice expressed lower levels of inflammatory markers (Nos2, Il6); however, NOX2-KO mice were hyperphagic and gained more weight than wild-type (WT) mice when fed either a chow or a high fat (HF) diet. Surprisingly, NOX2-KO mice stored less lipid in epididymal white adipose tissue but more lipid in liver, and had higher indices of liver inflammation and macrophage infiltration compared to WT mice. Contrary to our hypothesis, HF-fed NOX2-KO mice were hyperinsulinemic and more insulin resistant compared to HF-fed WT mice, likely as a result of their higher hepatic steatosis and inflammation. In summary, NOX2 depletion promoted hyperphagia, hepatic steatosis and inflammation with either normal or high fat-feeding, exacerbating insulin resistance. We propose that NOX2 participates in food intake control and lipid distribution in mice.

    April 22, 2014   doi: 10.1152/ajpendo.00089.2014   open full text
  • Improved cardiac function and dietary fatty acid metabolism after modest weight loss in subjects with impaired glucose tolerance.
    Labbe, S. M., Noll, C., Grenier-Larouche, T., Kunach, M., Bouffard, L., Phoenix, S., Guerin, B., Baillargeon, J.-P., Langlois, M.-F., Turcotte, E. E., Carpentier, A. C.
    AJP Endocrinology and Metabolism. April 22, 2014

    Using a novel positron emission tomography (PET) method with oral administration of 14(R,S)-[18F]-fluoro-6-thia-heptadecanoic acid (18FTHA), we recently demonstrated that subjects with impaired glucose tolerance (IGT) display an impairment in cardiac function associated with increased myocardial uptake of dietary fatty acids. Here, we determined whether modest weight loss induced by lifestyle changes might improve these cardiac metabolic and functional abnormalities. Nine participants with IGT, enrolled in a one-year lifestyle intervention trial, were invited to undergo determination of organ-specific postprandial dietary fatty acids partition using the oral 18FTHA method, and cardiac function and oxidative metabolic index using PET 11C-acetate kinetics with ECG-gated PET ventriculography prior to and after the intervention. The intervention resulted in significant weight loss and reduction of waist circumference, without significant change in postprandial plasma glucose, insulin, nonesterified fatty acid or triglyceride levels. We observed a significant increase in stroke volume, cardiac output and left ventricular ejection fraction associated with reduced myocardial oxidative metabolic index and fractional dietary fatty acid uptake. Modest weight loss corrects the exaggerated myocardial channelling of dietary fatty acids and improves myocardial energy substrate metabolism and function in IGT subjects ( NCT 00969007).

    April 22, 2014   doi: 10.1152/ajpendo.00638.2013   open full text
  • Acylcarnitines Activate Pro Inflammatory Signaling Pathways.
    Rutkowsky, J. M., Knotts, T. A., Ono-Moore, K. D., McCoin, C. S., Huang, S., Schneider, D., Singh, S., Adams, S. H., Hwang, D. H.
    AJP Endocrinology and Metabolism. April 22, 2014

    Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in type 2 diabetes and insulin resistance. In a previous study, we reported that mixed D, L isomers of C12- or C14-carnitine induced an NFB-luciferase reporter gene in RAW 264.7 cells suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical pro-inflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of COX-2 in a chain length dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25μM), used as a representative acylcarnitine, stimulated the expression and secretion of pro-inflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many pro-inflammatory signaling pathways including PRRs. Knockdown of MyD88, a key co-factor in PRR signaling and inflammation, blunted the pro-inflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking TLR2 and TLR4, and did not activate reporter constructs in TLR-overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

    April 22, 2014   doi: 10.1152/ajpendo.00656.2013   open full text
  • Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.
    Besedovsky, L., Linz, B., Dimitrov, S., Groch, S., Born, J., Lange, T.
    AJP Endocrinology and Metabolism. April 22, 2014

    Glucocorticoids are well-known to affect T-cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context with most of them employing synthetic glucocorticoids in rather non-physiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on 8 T-cell subpopulations in vivo and in vitro. Hydrocortisone (Cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T-cell subsets, with CD4+ and CD8+ naïve subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T-cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3+ and CD8+ T-cells appeared reduced following hydrocortisone infusion. However this was due to a selective extravasation of CD62L+ T-cell subsets, as hydrocortisone did neither affect CD62L expression on a subpopulation level nor did it affect CD62L expression in vitro. Corresponding results in opposite direction were observed after blocking endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T-cells to the bone marrow by up-regulating their CXCR4 expression, whereas its inhibiting effect on T-cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors.

    April 22, 2014   doi: 10.1152/ajpendo.00678.2013   open full text
  • Endothelial cells respond to hyperglycemia by increasing the LPL transporter GPIHBP1.
    Chiu, A. P.-L., Wang, F., Lal, N., Wang, Y., Zhang, D., Hussein, B., Wan, A., Vlodavsky, I., Rodrigues, B.
    AJP Endocrinology and Metabolism. April 15, 2014

    In diabetes, when glucose uptake and oxidation are impaired, the heart is compelled to use fatty acid (FA) almost exclusively for ATP. The vascular content of lipoprotein lipase (LPL), the rate-limiting enzyme that determines circulating triglyceride clearance, is largely responsible for this FA delivery, and increases following diabetes. Glycosylphosphatidylinositol anchored high density lipoprotein binding protein (GPIHBP1) [a protein abundantly expressed in the heart in endothelial cells (EC)] collects LPL from the interstitial space and transfers it across ECs onto the luminal binding sites of these cells, where the enzyme is functional. We tested whether EC respond to hyperglycemia by increasing GPIHBP1. Streptozotocin diabetes increased cardiac LPL activity and GPIHBP1 gene and protein expression. The increased LPL and GPIHBP1 were located at the capillary lumen. In vitro, passaging EC caused a loss of GPIHBP1, which could be induced on exposure to high glucose. The high-glucose-induced GPIHBP1 increased LPL shuttling across EC monolayers. GPIHBP1 expression was linked to the EC content of heparanase. Moreover, active heparanase increased GPIHBP1 gene and protein expression. Both EC and myocyte heparan sulfate proteoglycan (HSPG) bound platelet-derived growth factor (PDGF) released by heparanase caused augmentation of GPIHBP1. Overall, our data suggest that this protein "ensemble" (heparanase-PDGF-GPIHBP1) cooperates in the diabetic heart to regulate FA delivery and utilization by the cardiomyocytes. Interrupting this axis may be a novel therapeutic strategy to restore metabolic equilibrium, curb lipotoxicity, and help prevent or delay heart dysfunction characteristic of diabetes.

    April 15, 2014   doi: 10.1152/ajpendo.00007.2014   open full text
  • Long-lived crowded litter (CL) mice exhibit lasting effects on insulin sensitivity and energy homeostasis.
    Sadagurski, M., Landeryou, T., Blandino-Rosano, M., Cady, G., Elghazi, L., Meister, D., See, L., Bartke, A., Bernal-Mizrachi, E., Miller, R. A.
    AJP Endocrinology and Metabolism. April 15, 2014

    The action of nutrients on early postnatal growth can influence mammalian aging and longevity. Recent work has demonstrated that limiting nutrient availability in the first three weeks of life (by increasing the number of pups, in the crowded litter (CL) model) leads to extension of mean and maximal lifespan in genetically normal mice. In this study we aimed to characterize the impact of early life nutrient intervention on glucose metabolism and energy homeostasis in CL mice. In our study we used mice from litters supplemented to 12 or 15 pups and compared those to control litters limited to 8 pups. At weaning and then throughout adult life, CL mice are significantly leaner and consume more oxygen relative to control mice. At 6 months of age, CL mice had low fasting leptin concentrations, and low-dose leptin injections reduced body weight and food intake more in CL female mice than in controls. At 22 months, CL female mice also have smaller adipocytes compared to controls. Glucose and insulin tolerance tests show an increase in insulin sensitivity in 6 month old CL male mice, and females become more insulin sensitive later in life. Furthermore, β-cell mass was significantly reduced in the CL male mice and was associated with reduction in β-cell proliferation rate in these mice. Together, these data show that early life nutrient intervention has a significant lifelong effect on metabolic characteristics which may contribute to the increased lifespan of CL mice.

    April 15, 2014   doi: 10.1152/ajpendo.00031.2014   open full text
  • Glucose hypometabolism is highly localized but lower cortical thickness and brain atrophy are widespread in cognitively normal older adults.
    Nugent, S., Castellano, C.-A., Goffaux, P., Whittingstall, K., Lepage, M., Paquet, N., Bocti, C., Fulop, T., Cunnane, S. C.
    AJP Endocrinology and Metabolism. April 15, 2014

    Several studies suggest that glucose hypometabolism may be present in specific brain regions in cognitively normal older adults and could contribute to the risk of subsequent cognitive decline. However, certain methodological shortcomings including a lack of partial volume effect (PVE) correction or insufficient cognitive testing confound the interpretation of most studies on this topic. We combined 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) and magnetic resonance (MR) imaging to quantify cerebral metabolic rate of glucose (CMRg) as well as cortical volume and thickness in 43 anatomically-defined brain regions from a group of cognitively normal younger (25±3 y old; n=25) and older adults (71±9 y old; n=31). After correcting for PVE, we observed 11-17% lower CMRg in three specific brain regions of the older group - the superior frontal cortex, caudal middle frontal cortex, and the caudate (p≤0.01 false discovery rate-corrected). In the older group, cortical volumes and cortical thickness were 13-33% and 7-18% lower respectively in multiple brain regions (p≤0.01 FDR correction). There were no differences in CMRg between individuals who were or were not prescribed antihypertensive medication. There were no significant correlations between CMRg and cognitive performance or metabolic parameters measured in fasting plasma. We conclude that highly localized glucose hypometabolism and widespread cortical thinning and atrophy can be present in older adults who are cognitively normal as assessed using age-normed neuropsychological testing measures.

    April 15, 2014   doi: 10.1152/ajpendo.00067.2014   open full text
  • Effects of hyperandrogenemia and increased adiposity on reproductive and metabolic parameters in young adult female monkeys.
    McGee, W., Bishop, C., Pohl, C., Chang, J., Marshall, J. C., Pau, F. K. Y., Stouffer, R. L., Cameron, J. L.
    AJP Endocrinology and Metabolism. April 15, 2014

    Many patients with hyperandrogenemia are overweight or obese, which exacerbates morbidities associated with polycystic ovary syndrome (PCOS). To examine the ability of testosterone (T) to generate PCOS-like symptoms, monkeys received T or cholesterol (control) implants (n=6/group) beginning prepubertally. As previously reported, T-treated animals had increased neuroendocrine drive to the reproductive axis (increased LH pulse frequency) at 5 years, without remarkable changes in ovarian or metabolic features. To examine the combined effects of T and obesity, at 5.5 years (human equivalent age: 17 years), monkeys were placed on a high-calorie, high-fat diet typical of Western cultures (Western style diet; WSD), which increased body fat from <2% (pre-WSD) to 15-19% (14 months WSD). By 6 months on WSD, LH pulse frequency in the controls increased to that of T-treated animals, whereas LH pulse amplitude decreased in both groups and remained low. The numbers of antral follicles present during the early follicular phase increased in both groups on the WSD, but maximal follicular size decreased by 50%. During the late follicular phase, T-treated females had greater numbers of small antral follicles than controls. T-treated monkeys also had lower progesterone during the luteal phase of the menstrual cycle. Although fasting insulin did not vary between groups, T-treated animals had decreased insulin sensitivity after one year on WSD. Thus, while WSD consumption alone led to some features characteristic of PCOS, T+WSD caused a more severe phenotype with regards to insulin insensitivity, increased numbers of antral follicles at midcycle, and decreased circulating luteal phase progesterone levels.

    April 15, 2014   doi: 10.1152/ajpendo.00310.2013   open full text
  • Inhibition of connexin36 hemichannels by glucose contributes to the stimulation of insulin secretion.
    Pizarro-Delgado, J., Fasciani, I., Temperan, A., Romero, M., Gonzalez-Nieto, D., Alonso-Magdalena, P., Nualart-Marti, A., Estil'les, E., Paul, D. L., Martin-del-Rio, R., Montanya, E., Solsona, C., Nadal, A., Barrio, L. C., Tamarit-Rodriguez, J.
    AJP Endocrinology and Metabolism. April 15, 2014

    The existence of functional connexin36 (Cx36) hemichannels in β-cells was investigated in pancreatic islets of rat and wild-type (Cx36+/+), mono- (Cx36+/-), and bi-allelic (Cx36-/-) knockout mice. Hemichannel opening by KCl depolarization was studied measuring ATP release and changes of intracellular ATP (ADP). Cx36+/+ islets lost ATP after depolarization with 70 mM KCl at 5 mM glucose; ATP loss was prevented by 8 and 20 mM glucose or 50 µM mefloquine (connexin inhibitor). ATP content was higher in Cx36-/- than Cx36+/+ islets and was not decreased by KCl depolarization; Cx36+/- islets showed values between that of control and homozygous islets. 5 mM extracellular ATP increased ATP content, ATP/ADP ratio, and induced a biphasic insulin secretion in depolarized Cx36+/+ and Cx36+/- but not Cx36-/- islets. Cx36 hemichannels expressed in oocytes opened upon depolarization of membrane potential and their activation was inhibited by mefloquine and glucose (IC50~8 mM). It is postulated that glucose-induced inhibition of Cx36 hemichannels in islet β-cells might avoid depolarization induced ATP loss allowing an optimum increase of the ATP /ADP ratio by sugar metabolism and a biphasic stimulation of insulin secretion. Gradual suppression of glucose-induced insulin release in Cx36+/- and Cx36-/- islets confirms that Cx36 gap-junction channels are necessary for a full secretory stimulation and might account for the glucose intolerance observed in mice with defective Cx36 expression. Mefloquine targeting of Cx36 on both gap junctions and hemichannels also suppresses glucose-stimulated secretion. By contrast, glucose stimulation of insulin secretion requires Cx36 hemichannels' closure but keeping gap junction channels opened.

    April 15, 2014   doi: 10.1152/ajpendo.00358.2013   open full text
  • Hepatic mitochondrial and ER stress induced by defective PPAR{alpha} signaling in the pathogenesis of hepatic steatosis.
    Su, Q., Baker, C., Christian, P., Naples, M., Tong, X., Zhang, K., Santha, M., Adeli, K.
    AJP Endocrinology and Metabolism. April 15, 2014

    Emerging evidence demonstrates a close interplay between disturbances in mitochondrial function and ER homeostasis in the development of the metabolic syndrome. The present investigation sought to advance our understanding of the communication between mitochondrial dysfunction and ER stress in the onset of hepatic steatosis in male rodents with defective peroxisome proliferator-activated receptor (PPAR) α signaling. Genetic depletion of PPARα or perturbation of PPARα signaling by high-fructose diet compromised the functional activity of metabolic enzymes involved in mitochondrial fatty acid β-oxidation and induced hepatic mitochondrial stress in rats and mice. Inhibition of PPARα activity further enhanced expression of apolipoprotein B (apoB) mRNA and protein, which was associated with reduced mRNA expression of the sarco/endoplasmic reticulum calcium ATPase (SERCA), the induction of hepatic ER stress and hepatic steatosis. Restoration of PPARα activity recovered the metabolic function of the mitochondria and ER, alleviated systemic hypertriglyceridemia and improved hepatic steatosis. These findings unveil novel roles for PPARα in mediating stress signals between hepatic subcellular stress-responding machinery, and in the onset of hepatic steatosis under conditions of metabolic stress.

    April 15, 2014   doi: 10.1152/ajpendo.00438.2013   open full text
  • Effects of PYY3-36 and GLP-1 on energy intake, energy expenditure and appetite in overweight men.
    Schmidt, J. B., Gregersen, N. T., Pedersen, S. D., Arentoft, J. L., Ritz, C., Schwartz, T. W., Holst, J. J., Astrup, A., Sjodin, A.
    AJP Endocrinology and Metabolism. April 15, 2014

    Aim: To examine the effects of GLP-1 and PYY3-36, separately and in combination, on energy intake, energy expenditure, appetite sensations, glucose and fat metabolism, ghrelin and vital signs in healthy overweight men. Methods: 25 healthy, male subjects participated in this randomized, double-blinded, placebo-controlled 4-arm crossover study (BMI:29±3 kg/m2, age:33±9 years). On separate days they received a 150 min intravenous infusion of either a) 0.8pmol/kg/min PYY3-36, b) 1.0 pmol/kg/min GLP-1, c) a+b, or d) placebo. Ad libitum energy intake was assessed during the final 30 min. Measurements of appetite sensations, energy expenditure and fat oxidation, vital signs and blood variables were collected throughout the infusion period. Results: No effect on energy intake was found after monoinfusions of PYY3-36 (-4.2±4.8%, P=0.8) or GLP-1 (-3.0±4.5%, P=0.9). However, the co-infusion reduced energy intake compared to placebo (-30.4±6.5%, P<0.0001) and more than the sum of the monoinfusions (P<0.001), demonstrating a synergistic effect. Co-infusion slightly increased sensation of nausea (P<0.05), but this effect could not explain the effect on energy intake. A decrease in plasma ghrelin was found after all treatments compared to placebo (all P<0.05); however, infusions of GLP-1+PYY3-36 resulted in an additional decrease compared to the monoinfusions (both P<0.01). Conclusion: Co-infusion of GLP-1 and PYY3-36 exerted a synergistic effect on energy intake. The satiating effect of the meal was enhanced by GLP-1 and PYY3-36 in combination compared to placebo. Co-infusion was accompanied by slightly increased nausea and a decrease in plasma ghrelin, but neither of these factors could explain the reduction in energy intake.

    April 15, 2014   doi: 10.1152/ajpendo.00569.2013   open full text
  • Neuronostatin inhibits glucose-stimulated insulin secretion via a direct action on the pancreatic alpha cell.
    Salvatori, A. S., Elrick, M. M., Samson, W. K., Corbett, J. A., Yosten, G. L. C.
    AJP Endocrinology and Metabolism. April 15, 2014

    Neuronostatin is a recently described peptide hormone encoded by the somatostatin gene. We previously showed that intra-peritoneal injection of neuronostatin into mice resulted in c-Jun accumulation in pancreatic islets in a pattern consistent with the activation of glucagon-producing alpha cells. We therefore hypothesized that neuronostatin could influence glucose homeostasis via a direct effect on the alpha cell. Neuronostatin enhanced low glucose-induced glucagon release in isolated rat islets and in the immortalized alpha cell line, αTC1-9. Furthermore, incubation with neuronostatin led to an increase in transcription of glucagon mRNA, as determined by RT-PCR. Neuronostatin also inhibited glucose-stimulated insulin secretion from isolated islets. However, neuronostatin did not alter insulin release from the beta cell line INS 832/13, indicating that the effect of neuronostatin on insulin secretion may be secondary to a direct action on the alpha cell. In agreement with our in vitro data, intra-arterial infusion of neuronostatin in male rats delayed glucose disposal and inhibited insulin release during a glucose challenge. These studies suggest that neuronostatin participates in maintaining glucose homeostasis through cell-cell interactions between α-cells and β-cells in the endocrine pancreas leading to attenuation in insulin secretion.

    April 15, 2014   doi: 10.1152/ajpendo.00599.2013   open full text
  • Coinjection of CCK and leptin reduces food intake via increased CART/TRH and reduced AMPK phosphorylation in the hypothalamus.
    Akieda-Asai, S., Poleni, P.-E., Date, Y.
    AJP Endocrinology and Metabolism. April 15, 2014

    CCK and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important for elucidating the mechanisms by which energy balance is maintained. We found here that coadministration of subthreshold CCK and leptin, which individually have no effect on feeding, dramatically reduced food intake in rats. Phosphorylation of AMP-activated protein kinase (AMPK) in the hypothalamus significantly decreased after coinjection of CCK and leptin. In addition, coadministration of these hormones significantly increased mRNA levels of anorectic cocaine- and amphetamine-regulated transcript (CART) and TRH in the hypothalamus. The interactive effect of CCK and leptin on food intake was abolished by intracerebroventricular preadministration of the AMPK activator AICAR or anti-CART/anti-TRH antibodies. These findings indicate that coinjection of CCK and leptin reduces food intake via reduced AMPK phosphorylation and increased CART/TRH in the hypothalamus. Furthermore, by using midbrain-transected rats, we investigated the role of the neural pathway from the hindbrain to the hypothalamus in the interaction of CCK and leptin to reduce food intake. Food-intake reduction induced by coinjection of CCK and leptin was blocked in midbrain-transected rats. Therefore, the neural pathway from hindbrain to hypothalamus plays an important role in transmitting the anorectic signals provided by coinjection of CCK and leptin. Our findings give further insight into the mechanisms of feeding and energy balance.

    April 15, 2014   doi: 10.1152/ajpendo.00664.2013   open full text
  • Glucotoxicity targets hepatic glucokinase in Zucker diabetic fatty rats, a model of type 2 diabetes associated with obesity.
    Ueta, K., O'Brien, T. P., McCoy, G. A., Kim, K., Healey, E. C., Farmer, T. D., Donahue, E. P., Condren, A. B., Printz, R. L., Shiota, M.
    AJP Endocrinology and Metabolism. April 08, 2014

    A loss of glucose effectiveness to suppress hepatic glucose production as well as increase hepatic glucose uptake and storage as glycogen is associated with a defective increase in glucose phosphorylation catalyzed by glucokinase (GK) in Zucker diabetic fatty rats (ZDF). We extended these observations by investigating the role of persistent hyperglycemia (glucotoxicity) in the development of impaired hepatic GK activity in ZDF. We measured expression and localization of GK and GK-regulatory protein (GKRP), translocation of GK, and hepatic glucose flux in response to a gastric mixed meal load (MMT) and hyperglycemic-hyperinsulinemic clamp after 1 or 6 weeks of treatment with sodium-glucose transporter 2 inhibitor used to correct the persistent hyperglycemia of ZDF. Defective augmentation of glucose phosphorylation in response to a rise in plasma glucose in ZDF was associated with the co-residency of GKRP with GK in the cytoplasm in the mid stage of diabetes, which was followed by a decrease on GK protein levels due to impaired post-transcriptional processing in the late stage of diabetes. Correcting hyperglycemia from the middle diabetic stage normalized the rate of glucose phosphorylation by maintaining GK protein levels, restoring normal nuclear residency of GK and GKRP under basal condition and normalizing translocation of GK from the nucleus to the cytoplasm with GKRP remaining in the nucleus in response to a rise in plasma glucose. This improved the liver's metabolic inability to respond to hyperglycemic-hyperinsulinemia. Glucotoxicity is responsible for loss of glucose effectiveness on hepatic glucose flux by affecting GK regulation in the ZDF.

    April 08, 2014   doi: 10.1152/ajpendo.00507.2013   open full text
  • Inhibition of JNK by novel curcumin analog C66 prevents diabetic cardiomyopathy with a preservation of cardiac metallothionein expression.
    Wang, Y., Zhou, S., Sun, W., McClung, K., Pan, Y., Liang, Q., Tan, Y., Zhao, Y., Liu, Q., Sun, J., Cai, L.
    AJP Endocrinology and Metabolism. April 08, 2014

    The development of diabetic cardiomyopathy is attributed to diabetic oxidative stress, which may be related to the mitogen activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) activation. The present study tested a hypothesis whether curcumin analog, C66 [(2E,6E)-2,6-bis(2-(trifluoromethyl)benzylidene) cyclohexanone] as a potent antioxidant can protect diabetes-induced cardiac functional and pathogenic changes via inhibition of JNK function. Diabetes was induced with a single intraperitoneal injection of streptozotocin in male C57BL/6 mice. Diabetic and age-matched control mice were randomly divided into three groups, each group treated with C66, JNK inhibitor (JNKi, sp600125) or vehicle (1% CMC-Na solution) by gavage at 5mg/kg every other day for three months. Neither C66 nor JNKi impacted diabetic hyperglycemia and inhibition of body-weight gain, but both significantly prevented diabetes-induced JNK phosphorylation in the heart. Compared with basal line, cardiac function was significantly decreased in diabetic mice at 3 months of diabetes, but not in C66- or JNKi-treated diabetic mice. Cardiac fibrosis, oxidative damage, endoplasmic reticulum stress, and cell apoptosis, examined by Sirius-red staining, western blot, and thiobarbituric acid assay, were also significantly increased in diabetic mice, all which were prevented by C66 or JNKi treatment under diabetic conditions. Cardiac metallothionein expression was significantly decreased in diabetic mice, but almost normal in C66 or JNKi treated diabetic mice. These results suggest that like JNKi, C66, is able to prevent diabetic up-regulation of JNK function, resulting in a prevention of diabetes-induced cardiac fibrosis, oxidative stress, endoplasmic reticulum stress, and cell death, along with a preservation of cardiac metallothionein expression.

    April 08, 2014   doi: 10.1152/ajpendo.00629.2013   open full text
  • Mice Deleted for GPAT3 Have Reduced GPAT Activity in White Adipose Tissue and Altered Energy and Cholesterol Homeostasis in Diet-induced Obesity.
    Cao, J., Perez, S. M., Goodwin, B., Lin, Q., Peng, H., Qadri, A., Zhou, Y., Clark, R. W., Perreault, M., Tobin, J. F., Gimeno, R. E.
    AJP Endocrinology and Metabolism. April 08, 2014

    Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first step in the synthesis of glycerolipids and glycerophospholipids. Microsomal GPAT, the major GPAT activity, is encoded by at least two closely related genes, GPAT3 and GPAT4. To investigate the in vivo functions of GPAT3, we generated Gpat3-deficient mice (Gpat3-/-). Total GPAT activity in white adipose tissue of Gpat3-/- mice was reduced by 80%, suggesting that GPAT3 is the predominant GPAT in this tissue. In liver, GPAT3-deletion had no impact on total GPAT activity, but resulted in a 30% reduction in N-ethylmaleimide-sensitive GPAT activity. The Gpat3-/- mice were viable and fertile, and exhibited no obvious metabolic abnormalities on standard laboratory chow. However, when fed a high-fat diet, female Gpat3-/- mice showed decreased body weight gain and adiposity and increased energy expenditure. Increased energy expenditure was also observed in male Gpat3-/- mice, although it was not accompanied by a significant change in body weight. GPAT3-deficiency lowered fed, but not fasted, glucose levels, and tended to improve glucose tolerance in diet-induced obese male and female mice. On a high-fat diet, Gpat3-/- mice had enlarged livers and displayed a dysregulation in cholesterol metabolism. These data establish GPAT3 as the primary GPAT in white adipose tissue and reveal an important role of the enzyme in regulating energy, glucose, and lipid homeostasis.

    April 08, 2014   doi: 10.1152/ajpendo.00666.2013   open full text
  • C/EBP{alpha} Regulates Macrophage Activation and Systemic Metabolism.
    Lee, B., Qiao, L., Lu, M., Yoo, H. s., Cheung, W. W., Mak, R., Schaack, J., Feng, G.-S., Chi, N.-W., Olefsky, J. M., Shao, J.
    AJP Endocrinology and Metabolism. April 01, 2014

    Macrophage infiltration plays an important role in obesity-induced insulin resistance. CCAAT-enhancer-binding protein α (C/EBPα) is a transcription factor that is highly expressed in macrophages. To examine the roles of C/EBPα in regulating macrophage functions and energy homeostasis, macrophage-specific C/EBPα knock-out (MαKO) mice were created. Chow-fed MαKO mice exhibited higher body fat mass and decreased energy expenditure despite no change in food intake. However, the obese phenotype disappeared after high fat (HF) diet feeding. Although there was a transient decrease in insulin sensitivity of chow-fed young MαKO mice, systemic insulin sensitivity was protected during HF-feeding due to preserved insulin sensitivity in skeletal muscle. We also found that C/EBPα deficient macrophages exhibited a blunted response of cytokine-induced expression of M1 and M2 macrophage markers suggesting that C/EBPα controls both M1 and M2 polarization. Consistent with decreased exercise capacity, mitochondrial respiration rates and signal pathways for fatty acid oxidation were remarkably reduced in the skeletal muscle of chow-fed MαKO mice. Furthermore, expression levels of inflammatory cytokines were reduced in skeletal muscle of HF-fed MαKO mice. Together, these results implicate that C/EBPα is required for macrophage functions, which plays an important role in maintaining skeletal muscle energy metabolism.

    April 01, 2014   doi: 10.1152/ajpendo.00002.2014   open full text
  • Estrogen signaling prevents diet-induced hepatic insulin resistance in male mice with obesity.
    Zhu, L., Martinez, M. N., Emfinger, C. H., Palmisano, B. T., Stafford, J. M.
    AJP Endocrinology and Metabolism. April 01, 2014

    The development of insulin resistance in the liver is a key event that drives dyslipidemia, and predicts diabetes and cardiovascular risk with obesity. Clinical data show that estrogen signaling in males helps prevent adiposity and insulin resistance, which may be mediated through estrogen receptor α (ERα). The tissues and pathways that mediate the benefits of estrogen signaling in males with obesity are not well defined. In female mice, ERα signaling in the liver helps to correct pathway-selective insulin resistance with estrogen treatment after ovariectomy. We assessed the importance of liver estrogen signaling in males using liver ERα knockout (LKO) mice fed a high-fat diet (HFD). We found that the LKO male mice had decreased insulin sensitivity compared to their wild-type floxed (fl/fl) littermates during hyperinsulinemic-euglycemic clamps. Insulin failed to suppress endogenous glucose production in LKO mice, indicating liver insulin resistance. Insulin promoted glucose disappearance in LKO and fl/fl mice similarly. In the liver, insulin failed to induce phosphorylation of AKT-Ser473 and exclude FOXO1 from the nucleus in LKO mice, a pathway important for liver glucose and lipid metabolism. Liver triglycerides and diacylglycerides were also increased in LKO mice, which corresponded with dysregulation of insulin stimulated ACC phosphorylation and DGAT1/2 protein levels. Our studies demonstrate that estrogen signaling through ERα in the liver helps prevent whole-body and hepatic insulin resistance associated with HFD-feeding in males. Augmenting hepatic estrogen signaling through ERα may lessen the impact of obesity on diabetes and cardiovascular risk in males.

    April 01, 2014   doi: 10.1152/ajpendo.00579.2013   open full text
  • Activation of mTORC1 Signaling and Protein Synthesis in Human Muscle Following Blood Flow Restriction Exercise is Inhibited by Rapamycin.
    Gundermann, D. M., Walker, D. K., Reidy, P. T., Borack, M. S., Dickinson, J. M., Volpi, E., Rasmussen, B. B.
    AJP Endocrinology and Metabolism. April 01, 2014

    Restriction of blood flow to a contracting muscle during low-intensity resistance exercise (BFR exercise) stimulates mTORC1 signaling and protein synthesis in human muscle within 3 hours post-exercise. However, there is a lack of mechanistic data to provide a direct link between mTORC1 activation and protein synthesis in human skeletal muscle following BFR exercise. Therefore, the primary purpose of this study was to determine if mTORC1 signaling is necessary for stimulating muscle protein synthesis after BFR exercise. A secondary aim was to describe the 24-hour time-course response in muscle protein synthesis and breakdown following BFR exercise. Sixteen healthy young men were randomized to one of two groups. Both the control group (CON) and rapamycin (RAP) groups completed BFR exercise however, RAP was administered 16mg of the mTOR inhibitor, rapamycin, one hour prior to BFR exercise. BFR exercise consisted of 4 sets of leg extension exercise at 20% of 1RM. Muscle biopsies were collected from the vastus lateralis before exercise and at 3, 6 and 24 hr after BFR exercise. Mixed muscle protein fractional synthetic rate increased by 42% at 3 hr post-exercise and 69% at 24 hr post-exercise in CON whereas this increase was inhibited in the RAP group. Phosphorylation of mTOR (Ser2448) and S6K1 (Thr389) were also increased in CON but inhibited in RAP. Mixed muscle protein breakdown was not significantly different across time or groups. We conclude that activation of mTORC1 signaling and protein synthesis in human muscle following BFR exercise is inhibited in the presence of rapamycin.

    April 01, 2014   doi: 10.1152/ajpendo.00600.2013   open full text
  • Adipose tissue insulin resistance due to loss of PI3K p110{alpha} leads to decreased energy expenditure and obesity.
    Nelson, V. L. B., Jiang, Y.-P., Dickman, K. G., Ballou, L. M., Lin, R. Z.
    AJP Endocrinology and Metabolism. April 01, 2014

    Adipose tissue is a highly insulin responsive organ that contributes to metabolic regulation. Insulin resistance in the adipose tissue affects systemic lipid and glucose homeostasis. Phosphoinositide 3-kinase (PI3K) mediates downstream insulin signaling in adipose tissue, but its physiological role in vivo remains unclear. Using Cre recombinase driven by the aP2 promoter, we created mice that lack the class 1A PI3K catalytic subunit p110α or p110β specifically in the white and brown adipose tissue. The loss of p110α, not p110β, resulted in increased adiposity, glucose intolerance and liver steatosis. Mice lacking p110α in adipose tissue exhibited a decrease in energy expenditure but no change in food intake or activity as compared to control animals. This low energy expenditure is a consequence of low cellular respiration in the brown adipocytes caused by a decrease in expression of key mitochondrial genes including uncoupling protein-1. These results illustrate a critical role of p110α in the regulation of energy expenditure through modulation of cellular respiration in the brown adipose tissue and suggest that compromised insulin signaling in adipose tissue might be involved in the onset of obesity.

    April 01, 2014   doi: 10.1152/ajpendo.00625.2013   open full text
  • The order of concurrent endurance and resistance exercise modifies mTOR signaling and protein synthesis in rat skeletal muscle.
    Ogasawara, R., Sato, K., Matsutani, K., Nakazato, K., Fujita, S.
    AJP Endocrinology and Metabolism. April 01, 2014

    Concurrent training, a combination of endurance (EE) and resistance exercise (RE) performed in succession, may compromise the muscle hypertrophic adaptations induced by RE alone. However, little is known about the molecular signaling interactions underlying the changes in skeletal muscle adaptation during concurrent training. Here, we used an animal model to investigate whether EE before or after RE affects the molecular signaling associated with muscle protein synthesis, specifically the interaction between RE-induced mammalian target of rapamycin complex 1 (mTORC1) signaling and EE-induced AMP-activated protein kinase (AMPK) signaling. Male Sprague-Dawley rats were divided into 5 groups: an EE (treadmill, 25 m/min, 60 min) group, an RE (maximum isometric contraction via percutaneous electrical stimulation for 3 s x 10, 5 sets) group, an EE before RE group, an EE after RE group, and a non-exercise control (CON) group. Phosphorylation of p70S6K, a marker of mTORC1 activity, was significantly increased 3 h after RE in both the EE before RE and EE after RE groups, but the increase was smaller in latter. Further, protein synthesis was greatly increased 6 h after RE in the EE before RE group. Increases in the phosphorylation of AMPK and Raptor were observed only in the EE after RE group. Akt and mTOR phosphorylation were increased in both groups with no between-group differences. Our results suggest that the last bout of exercise dictates the molecular responses, and that mTORC1 signaling induced by any prior bout of RE may be downregulated by a subsequent bout of EE.

    April 01, 2014   doi: 10.1152/ajpendo.00647.2013   open full text
  • Insulin Sensitivity Index in Type 1 Diabetes and Following Human Islet Transplantation: Comparison of the Minimal Model to Euglycemic Clamp Measures.
    Rickels, M. R., Kong, S. M., Fuller, C., Dalton-Bakes, C., Ferguson, J. F., Reilly, M. P., Teff, K. L., Naji, A.
    AJP Endocrinology and Metabolism. April 01, 2014

    Insulin sensitivity is impaired in type 1 diabetes (T1D) and may be enhanced by islet transplantation, an effect best explained by improved metabolic control. While the minimal model index of insulin sensitivity, SI, has been used in studies of T1D, it has not before been evaluated against gold-standard measures derived from the euglycemic clamp. We sought to determine how well minimal model SI derived from an insulin-modified frequently-sampled intravenous glucose tolerance (FSIGT) test compared to total body and peripheral insulin sensitivity estimates derived from the hyperinsulinemic euglycemic clamp in subjects with T1D and following islet transplantation. Twenty-one T1D subjects were evaluated, including a subgroup (n=12) studied again after intrahepatic islet transplantation, with results compared to normal controls (n=11 for the FSIGT). The transplant recipients received 9648±666 islet equivalents/kg with reduction in HbA1c from 7.1±0.2 to 5.5±0.1% (P<0.01) and 10/12 were insulin-independent. FSIGT derived SI was reduced in T1D pre- compared to post-transplant and to normal (1.76±0.45 vs. 4.21±0.34 vs. 4.45 ±0.81 x10-4(μU/ml)-1•min-1; P<0.01 for both). Similarly, clamp derived total body, and by the isotopic dilution method with 6,6-2H2-glucose, peripheral insulin sensitivity increased in T1D from pre- to post-transplant (P<0.05 for both). The predictive power (r2) between volume corrected SIC and measures of total and peripheral insulin sensitivity was 0.66 and 0.70 respectively (P<0.00001 for both). That the minimal model SIC is highly correlated to the clamp derived measures indicates that the FSIGT is an appropriate methodology for the determination of insulin sensitivity in T1D and following islet transplantation.

    April 01, 2014   doi: 10.1152/ajpendo.00667.2013   open full text
  • Maternal high fat diet induces insulin resistance and deterioration of pancreatic {beta} cell function in adult offspring with gender differences in mice.
    Yokomizo, H., Inoguchi, T., Sonoda, N., Sakaki, Y., Maeda, Y., Inoue, T., Hirata, E., Takei, R., Ikeda, N., Fujii, M., Fukuda, K., Sasaki, H., Takayanagi, R.
    AJP Endocrinology and Metabolism. April 01, 2014

    Intrauterine environment may influence the health of postnatal offspring. There have been many studies on the effects of maternal high fat diet (HFD) on diabetes and glucose metabolism in offspring. Here, we investigated the effects in male and female offspring. C57/BL6J mice were bred and fed either control diet (CD) or HFD from conception to weaning, and offspring were fed CD or HFD from 6 to 20 weeks. At 20 weeks, maternal HFD induced glucose intolerance and insulin resistance in offspring. Additionally, liver triacylglycerol content, adipose tissue mass and inflammation increased in maternal HFD. In contrast, extending previous observations, insulin secretion at glucose tolerance test, islet area, insulin content and PDX-1 mRNA levels in isolated islets were lower in maternal HFD in male, while they were higher in female. Oxidative stress in islets increased in maternal HFD in male, while there were no differences in female. Plasma estradiol levels were lower in male than in female, and decreased in offspring fed with HFD, and also decreased by maternal HFD, suggesting that females may be protected from insulin deficiency by inhibiting oxidative stress. In conclusion, maternal HFD induced insulin resistance and deterioration of pancreatic β cell function with marked gender differences in adult offspring, accompanied by adipose tissue inflammation and liver steatosis. Additionally, our results demonstrate that potential mechanisms underlying gender differences in pancreatic β cell function may be partially related to increases in oxidative stress in male islets and decreased plasma estradiol levels in male.

    April 01, 2014   doi: 10.1152/ajpendo.00688.2013   open full text
  • Fatty Acid Synthase is Required for Mammary Gland Development and Milk Production during Lactation.
    Suburu, J., Shi, L., Wu, J., Wang, S., Samuel, M., Thomas, M. J., Kock, N. D., Yang, G., Kridel, S., Chen, Y. Q.
    AJP Endocrinology and Metabolism. March 25, 2014

    The mammary gland is one of few adult tissues that strongly induce de novo fatty acid synthesis upon physiological stimulation, suggesting that fatty acid is important for milk production during lactation. The committed enzyme to perform this function is fatty acid synthase (FASN). To determine whether de novo fatty acid synthesis is obligatory or dietary fat is sufficient for mammary gland development and function during lactation, Fasn was specifically knocked out in mouse mammary epithelial cells. We found that deletion of Fasn hindered the development and induced premature involution of the lactating mammary gland, significantly decreased short- and medium-chain fatty acids and total fatty acid contents in the milk. Consequently, pups nursing from Fasn knockout mothers experienced growth retardation and pre-weanling death, which was rescued by cross-fostering pups to a lactating wild type mother. These results demonstrate that FASN is essential for the development, functional competence, and maintenance of the lactating mammary gland.

    March 25, 2014   doi: 10.1152/ajpendo.00514.2013   open full text
  • Interaction between dietary fat and exercise on excess post-exercise oxygen consumption (EPOC).
    Frost, E. A., Redman, L. M., de Jonge, L., Rood, J. C., Zachwieja, J. J., Volaufova, J., Bray, G. A., Smith, S. R.
    AJP Endocrinology and Metabolism. March 18, 2014

    The objective of this study was to determine the effect of increased physical activity on subsequent sleeping energy expenditure (SEE) measured in a whole room calorimeter under differing levels of dietary fat. We hypothesized that increased physical activity would increase SEE. Six healthy, young men participated in a randomized, single blind, crossover study. Subjects repeated an eight-day protocol under four conditions separated by at least 7 days. During each condition, subjects consumed an isoenergetic diet consisting of 37% fat, 15% protein and 48% carbohydrate for the first four days and for the following four days, SEE and energy balance were measured in a respiration chamber. The first chamber day served as a baseline measurement and for the remaining three days, diet and activity were randomly assigned as high fat/exercise, high fat/sedentary, low fat/exercise, or low fat/sedentary. Energy balance was not different between conditions. When the dietary fat was increased to 50%, SEE increased by 7.4% during exercise (P<0.05) relative to being sedentary (baseline day) but SEE did not increase with exercise when fat was lowered to 20%. SEE did not change when dietary fat was manipulated under sedentary conditions. Physical activity causes an increase in SEE when dietary fat is high (50%), but not when dietary fat is low (20%). Dietary fat content influences the impact of post-exercise induced increases in sleeping energy expenditure. This finding may help explain the conflicting data regarding the effect of exercise on energy expenditure.

    March 18, 2014   doi: 10.1152/ajpendo.00383.2013   open full text
  • Two weeks of metformin treatment induces AMPK dependent enhancement of insulin-stimulated glucose uptake in mouse soleus muscle.
    Kristensen, J. M., Treebak, J. T., Schjerling, P., Goodyear, L., Wojtaszewski, J. F. P.
    AJP Endocrinology and Metabolism. March 18, 2014

    Background: Metformin-induced activation of AMPK has been associated with enhanced glucose uptake in skeletal muscle but so far no direct causality has been examined. We hypothesized that an effect of in vivo metformin treatment on glucose uptake in mouse skeletal muscles is dependent upon AMPK signaling. Methods: Oral doses of metformin or saline treatment were given muscle-specific kinase α2 dead AMPK mice (KD) and wild type (WT) littermates either once or chronically for 2 weeks. Soleus and Extensor Digitorum Longus (EDL) muscles were used for measurements of glucose transport and Western blot analyzes. Results: Chronic treatment with metformin enhanced insulin-stimulated glucose uptake in soleus muscles of WT (45%, P<0.01), but not in AMPK KD mice. Insulin signaling at the level of Akt protein expression or Thr308 and Ser473 phosphorylation was not changed by metformin treatment. Downstream of Akt, TBC1D4 Thr642 and Ser711 phosphorylation and Rab4 and GLUT4 protein expressions were not affected by metformin treatment. Also AMPK catalytic subunit, TBC1D4 and hexokinase II proteins were unaltered after treatment. The acute metformin treatment did not affect glucose uptake in muscle of either of the genotypes. Conclusion: We provide novel evidence for a role of AMPK in potentiating the effect of insulin on glucose uptake in skeletal muscle in response to chronic metformin treatment.

    March 18, 2014   doi: 10.1152/ajpendo.00417.2013   open full text
  • A single intake of capsiate improves mechanical performance and bioenergetics efficiency in contracting mouse skeletal muscle.
    Kazuya, Y., Tonson, A., Pecchi, E., Dalmasso, C., Vilmen, C., Le Fur, Y., Bernard, M., Bendahan, D., Giannesini, B.
    AJP Endocrinology and Metabolism. March 18, 2014

    Capsiate is known to increase whole body oxygen consumption possibly via the activation of uncoupling processes, but its effect at skeletal muscle level remains poorly documented and conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated in mice, 2 hours after a single intake of either vehicle (control) or purified capsiate (at 10- or 100-mg/kg body weight), through a multidisciplinary approach combining in vivo and in vitro measurements. Mechanical performance and energy pathway fluxes were accessed strictly noninvasively during a standardized electrostimulation-induced exercise using an original device implementing 31-phosphorus magnetic resonance spectroscopy, and mitochondrial respiration was evaluated in isolated saponin-permeabilized fibers. Compared to control, both capsiate doses produced quantitatively similar effects at the energy metabolism level, including an about 2-fold decrease of the mitochondrial respiration sensitivity for ADP. Interestingly, they did alter neither oxidative phosphorylation nor uncoupling protein 3 gene expression at rest. During 6-min of maximal repeated isometric contractions, both doses reduced the amount of ATP produced from glycolysis and oxidative phosphorylation, but increased the relative contribution of oxidative phosphorylation to total energy turnover (+28% and +21% in 10-mg and 100-mg groups, respectively). ATP cost of contraction was further reduced in 10-mg (-35%) and 100-mg (-45%) groups. Besides, the highest dose also increased the force-generating capacity. These data present capsiate as a helpful candidate to enhance both muscle performance and oxidative phosphorylation during exercise, which could constitute a nutritional approach for improving health and preventing obesity and associated metabolic disorders.

    March 18, 2014   doi: 10.1152/ajpendo.00520.2013   open full text
  • The role of AMPK and PPAR{gamma}1 in exercise-induced lipoprotein lipase in skeletal muscle.
    Sasaki, T., Nakata, R., Inoue, H., Shimizu, M., INOUE, J., Sato, R.
    AJP Endocrinology and Metabolism. March 18, 2014

    Exercise can effectively ameliorate type 2 diabetes and insulin resistance. Here we show that the mRNA levels of one of peroxisome proliferator-activated receptor (PPAR) family members, PPAR1, and genes related to energy metabolism, including PPAR coactivator-1 protein α (PGC-1α) and lipoprotein lipase (LPL), increased in the gastrocnemius muscle of habitual exercise-trained mice. When mice were intraperitoneally administered an AMP-activated protein kinase (AMPK) activator 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), the mRNA levels of the aforementioned three genes increased in gastrocnemius muscle. AICAR treatment to C2C12 differentiated myotubes also increased PPAR1 mRNA levels, but not PPARα and mRNA levels, concomitant with increased PGC-1α mRNA levels. An AMPK inhibitor, compound C, blocked these AICAR effects. AICAR treatment increased the half-life of PPAR1 mRNA by nearly 3-fold (4 h - 12 h) by activating AMPK. When C2C12 myoblast cells infected with a PPAR1 expression lentivirus were differentiated into myotubes, PPAR1 overexpression dramatically increased LPL mRNA levels by more than 40-fold. In contrast, when PPAR1 expression was suppressed in C2C12 myotubes, LPL mRNA levels significantly reduced and the effect of AICAR on increased LPL gene expression was almost completely blocked. These results indicated that PPAR1 was intimately involved in LPL gene expression in skeletal muscle and the AMPK-PPAR1 pathway may play a role in exercise-induced LPL expression. Thus, we identified a novel critical role for PPAR1 in response to AMPK activation for controlling the expression of a subset of genes associated with metabolic regulation in skeletal muscle.

    March 18, 2014   doi: 10.1152/ajpendo.00691.2013   open full text
  • GTP Cyclohydrolase I Prevents Diabetic Impaired Endothelial Progenitor Cells and Wound Healing by Suppressing Oxidative Stress/Thrombospondin-1.
    Tie, L., Chen, L.-Y., Chen, D.-D., Xie, H.-H., Channon, K. M., Chen, A. F.
    AJP Endocrinology and Metabolism. March 18, 2014

    Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic refractory wounds. Endothelial nitric oxide synthase (eNOS), which critically regulates the mobilization and function of EPCs, is uncoupled in diabetes due to decreased cofactor tetrahydrobiopterin (BH4). We tested whether GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 synthesis, preserves EPC function in type 1 diabetic mice. Type 1 diabetes was induced in wild-type (WT) and GTPCH I transgenic (Tg-GCH) mice by intraperitoneal injection of streptozotocin (STZ). EPCs were isolated from the peripheral blood and bone marrow of WT, Tg-GCH, and GTPCH I-deficient hph-1 mice. The number of EPCs was significantly lower in STZ-WT mice and hph-1 mice, and was rescued in STZ Tg-GCH mice. Furthermore, GTPCH I overexpression improved impaired diabetic EPC migration and tube formation. EPCs from WT, Tg-GCH, and STZ-Tg-GCH mice were administered to diabetic excisional wounds, significantly accelerated wound healing, with a concomitant augmentation of angiogenesis. Flow cytometry measurements showed that intracellular nitric oxide (NO) levels were significantly reduced in STZ-WT mice and hph-1 mice, paralleled by increased superoxide anion level; both were rescued in STZ-Tg-GCH mice. Western blot analysis revealed that thrombospondin-1 (TSP-1) was significantly upregulated in the EPCs of STZ-WT mice and hph-1 mice, and suppressed in STZ-treated Tg-GCH mice. Our results demonstrate that the GTPCH I/BH4 pathway is critical to preserve EPC quantity, function, and regenerative capacity during wound healing in type 1 diabetic mice, at least partly, through the attenuation of superoxide and TSP-1 levels and augmentation of NO level.

    March 18, 2014   doi: 10.1152/ajpendo.00696.2013   open full text
  • Exercise as an intervention to improve metabolic outcomes after intrauterine growth restriction.
    Gatford, K. L., Kaur, G., Falcao-Tebas, F., Wadley, G. D., Wlodek, M. E., Laker, R. C., Ebeling, P. R., McConell, G. K.
    AJP Endocrinology and Metabolism. March 11, 2014

    Individuals born after intrauterine growth restriction (IUGR) are at an increased risk of developing diabetes in their adult life. IUGR impairs β-cell function and reduces β-cell mass thereby diminishing insulin secretion. IUGR also induces insulin resistance, with impaired insulin signaling in muscle in adult humans who were small for gestational age (SGA) and in rodent models of IUGR. There is epidemiological evidence in humans that exercise in adults can reduce the risk of metabolic disease following IUGR. However, it is not clear whether adult IUGR individuals benefit to the same extent from exercise as do normal birth weight individuals, as our rat studies suggest less of a benefit in those born IUGR. Importantly, however, there is some evidence from studies in rats that exercise in early life might be able to reverse or reprogram the long-term metabolic effects of IUGR. Studies are needed to address gaps in current knowledge, including determining the mechanisms involved in the reprogramming effects of early exercise in rats, whether exercise early in life or in adulthood has similar beneficial metabolic effects in larger animal models in which insulin resistance develops after IUGR. Human studies are also needed to determine whether exercise training improves insulin secretion and insulin sensitivity to the same extent in IUGR adults as in as control populations. Such investigations will have implications for customising the recommended level and timing of exercise to improve metabolic health after IUGR.

    March 11, 2014   doi: 10.1152/ajpendo.00456.2013   open full text
  • Loss of HIF-1{alpha} impairs GLUT4 translocation and glucose uptake by the skeletal muscle cells.
    Sakagami, H., Makino, Y., Mizumoto, K., Isoe, T., Takeda, Y., Watanabe, J., Fujita, Y., Takiyama, Y., Abiko, A., Haneda, M.
    AJP Endocrinology and Metabolism. March 11, 2014

    Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. Molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1α in homeostatic response to various cellular stimuli including insulin under normoxic condition. Thus we hypothesized HIF-1α is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12 myocytes in which HIF-1α is knocked-down by shRNA and examined intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1α expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the Akt substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMPK activation were abrogated as well. In addition, expression of constitutively-active mutant of HIF-1α (CA-HIF-1α) or upregulation of endogenous HIF-1α in C2C12 cells shows AS160 phosphorylation comparable to insulin stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1α. Taken together, HIF-1α is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in e.g. type 2 diabetes.

    March 11, 2014   doi: 10.1152/ajpendo.00597.2012   open full text
  • CXCL1/CXCL8 (GRO{alpha}/IL-8) in Human Diabetic Ketoacidosis Plasma Facilitate Leukocyte Recruitment to Cerebrovascular Endothelium In Vitro.
    Omatsu, T., Cepinskas, G., Clason, C., Patterson, E., Alharfi, I., Summers, K., Couraud, P.-O., Romero, I., Weksler, B., Fraser, D.
    AJP Endocrinology and Metabolism. March 11, 2014

    Diabetic ketoacidosis (DKA) in children is associated with intracranial vascular complications, possibly due to leukocyte-endothelial interactions. Our aim was to determine if DKA-induced inflammation promoted leukocyte adhesion to activated human cerebrovascular endothelium. Plasma was obtained from children with type-1 diabetes, either in acute DKA or in an insulin-controlled state (CON). Plasma concentrations of 21 inflammatory analytes were compared between groups. DKA was associated with altered circulating levels of CXCL1 (GROα), CXCL8 (IL-8), IL-6, IFNα2 and CXCL10 (IP-10), as compared to CON. These plasma analyte measurements were then used to create physiologically-relevant cytokine-mixtures (CM). Human cerebral microvascular endothelial cells (hCMEC/D3) were stimulated with either plasma (DKA-P or CON-P) or CM (DKA-CM or CON-CM) and assessed for polymorphonuclear neutrophil (PMN) adhesion. Stimulation of hCMEC/D3 with DKA-P or DKA-CM increased PMN adhesion to hCMEC/D3 under "flow" conditions. PMN adhesion to hCMEC/D3 was suppressed with neutralizing antibodies to CXCL1/CXCL8 or their hCMEC/D3 receptors CXCR1/CXCR2. DKA-P, but not DKA-CM, initiated oxidative stress in hCMEC/D3. Expression of ICAM-1, VCAM-1 and E-selectin were unaltered on hCMEC/D3 by either DKA-P or DKA-CM. In summary, DKA elicits inflammation in children associated with changes in circulating cytokines/chemokines. Increased CXCL1/CXCL8 instigated PMN adhesion to hCMEC/D3, possibly contributing to DKA-associated intracranial vascular complications.

    March 11, 2014   doi: 10.1152/ajpendo.00659.2013   open full text
  • PPAR{gamma} activation attenuates glucose intolerance induced by mTOR inhibition with rapamycin in rats.
    Festuccia, W. T., Blanchard, P.-G., Belchior, T., Chimin, P., Paschoal, V. A., Magdalon, J., Hirabara, S. M., Simoes, D., St-Pierre, P., Carpinelli, A. R., Marette, A., Deshaies, Y.
    AJP Endocrinology and Metabolism. March 11, 2014

    mTOR inhibition with rapamycin induces a diabetes-like syndrome characterized by severe glucose intolerance, hyperinsulinemia and hypertriglyceridemia, which are due to increased hepatic glucose production as well as reduced skeletal muscle glucose uptake and adipose tissue PPAR activity. Herein we tested the hypothesis that pharmacological PPAR activation attenuates the diabetes-like syndrome associated with chronic mTOR inhibition. Rats treated with the mTOR inhibitor rapamycin (2 mg/kg/day) in combination or not with the PPAR ligand rosiglitazone (15 mg/kg/day) for 15 days were evaluated for insulin secretion, glucose, insulin and pyruvate tolerances, skeletal muscle and adipose tissue glucose uptake and insulin signaling. Rosiglitazone corrected fasting hyperglycemia, attenuated the glucose and insulin intolerances and abolished the increase in fasting plasma insulin and C-peptide levels induced by rapamycin. Surprisingly, rosiglitazone markedly increased the plasma insulin and C-peptide responses to refeeding in rapamycin-treated rats. Furthermore, rosiglitazone partially attenuated rapamycin-induced gluconeogenesis as evidenced by the improved pyruvate tolerance and reduced mRNA levels of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase. Rosiglitazone also restored insulin's ability to stimulate glucose uptake and its incorporation into glycogen in skeletal muscle of rapamycin-treated rats, which was associated with normalization of Akt Ser473 phosphorylation. The rapamycin-mediated impairments of adipose tissue glucose uptake and incorporation into triacylglycerol, however, were unaffected by rosiglitazone. Our findings indicate that PPAR activation ameliorates some of the disturbances in glucose homeostasis and insulin action associated with chronic rapamycin treatment by reducing gluconeogenesis and insulin secretion and restoring muscle insulin signaling and glucose uptake.

    March 11, 2014   doi: 10.1152/ajpendo.00683.2013   open full text
  • Pro-inflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress and dynamics.
    Hahn, W. S., Kuzmicic, J., Burrill, J. S., Donoghue, M. A., Foncea, R., Jensen, M. D., Lavandero, S., Arriaga, E. A., Bernlohr, D. A.
    AJP Endocrinology and Metabolism. March 04, 2014

    Macrophage infiltration of adipose tissue and the chronic low-grade production of inflammatory cytokines have been mechanistically linked to the development of insulin resistance, the forerunner of type 2 diabetes mellitus. In this study we evaluated the chronic effects of TNFα, IL-6 and IL-1β on adipocyte mitochondrial metabolism and morphology using the 3T3-L1 model cell system. TNFα treatment of cultured adipocytes led to significant changes in mitochondrial bioenergetics including increased proton leak, decreased membrane potential, increased basal respiration and decreased ATP turnover. In contrast, while IL-6 and IL-1β decreased maximal respiratory capacity, they had no effect on membrane potential and varied effects on ATP turnover, proton leak and basal respiration. Only TNFα treatment of 3T3-L1 cells led to an increase in oxidative stress (as measured by superoxide anion production and protein carbonylation) and C16 ceramide synthesis. Treatment of 3T3-L1 adipocytes with cytokines led to decreased mRNA expression of key transcription factors and control proteins implicated in mitochondrial biogenesis including PGC1α and eNOS as well as deceased expression of COXIV and Cyt CCyt C. Whereas each cytokine led to effects on expression of mitochondrial markers, TNFα exclusively led to mitochondrial fragmentation, decreased the total level of Opa1 while increasing Opa1 cleavage without affecting expression of levels of mitofusin 2, DRP1 or mitofilin. In sum, these results indicate that inflammatory cytokines have unique and specialized effects on adipocyte metabolism but each leads to decreased mitochondrial function and a re-programming of fat cell biology.

    March 04, 2014   doi: 10.1152/ajpendo.00422.2013   open full text
  • Increased Myocardial Ischemia/reperfusion Injury in Renal Failure Involves Cardiac Adiponectin Signal Deficiency.
    Song, Y., Yu, Q., zhang, J., Huang, W., Liu, Y., Pei, H., Liu, J., Sun, L., Yang, L., Li, C., Li, Y., Zhang, F., Tao, L.
    AJP Endocrinology and Metabolism. March 04, 2014

    Plasma levels of adiponectin (APN) are significantly increased in patients with renal dysfunction and are inversely related to the risk of cardiovascular mortality. The present study was designed to determine the role of APN in myocardial ischemia/reperfusion (MI/R) injury in mice with renal failure, and delineate the underlying mechanisms. Renal failure was induced by subtotal nephrectomy (SN). Human recombinant globular domain of adiponectin (gAd) or full length adiponectin (fAd) was administered via intraperitoneal injection once daily for 7 consecutive days after SN and in vivo MI/R was introduced 3 weeks later. Both plasma and urinary levels of APN increased significantly in SN mice. Compared with sham-operated mice, cardiac function was significantly depressed and myocardial infarct size and apoptosis increased in SN mice following MI/R. The aggravated MI/R injury was further intensified in APN knockout mice and markedly ameliorated by treatment with gAd but not fAd. Moreover, SN increased myocardial NO metabolites, superoxide and their cytotoxic reaction product peroxynitrite, upregulated inducible NO synthase expression and decreased endothelial NOS phosphorylation. In addition, SN mice also exhibited reduced APN receptor-1 (AdipoR1) expression and AMPK activation. All these changes were further amplified in the absence of APN but reversed by gAd treatment. The present study demonstrates that renal dysfunction increases cardiac susceptibility to ischemic/reperfusion injury, which is associated with downregulated APN/AdipoR1/AMPK signaling and increased oxidative/nitrative stress in local myocardium, and provides the first evidence for the protective role of exogenous supplement of gAd on MI/R outcomes in renal failure.

    March 04, 2014   doi: 10.1152/ajpendo.00428.2013   open full text
  • Influence of aerobic exercise intensity on myofibrillar and mitochondrial protein synthesis in young men during early and late post-exercise recovery.
    Di Donato, D. M., West, D. W. D., Churchward-Venne, T. A., Breen, L., Baker, S. K., Phillips, S. M.
    AJP Endocrinology and Metabolism. March 04, 2014

    Aerobic exercise is typically associated with expansion of the mitochondrial protein pool and improvements in muscle oxidative capacity. The impact of aerobic exercise intensity on the synthesis of specific skeletal muscle protein sub-fractions is not known. We aimed to study the effect of aerobic exercise intensity on rates of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis over an early (0.5-4.5 h) and late (24-28 h) period during post-exercise. Using a within subject crossover design, eight males (21 ± 1 years, VO2 peak: 46.7 ± 2.0 mL•kg-1•min-1) performed two work-matched cycle ergometry exercise trials (LOW: 60 min at 30% Wmax; HIGH: 30 min at 60% Wmax) in the fasted state while undergoing a primed constant infusion of L-[ring-13C6]phenylalanine. Muscle biopsies were obtained at rest, and 0.5, 4.5, 24, and 28 h post-exercise to determine both the 'early' and 'late' response of MyoPS and MitoPS and the phosphorylation status of select proteins within both the Akt/mTOR and MAPK pathways. Over 24-28 h post-exercise, MitoPS was significantly greater after the HIGH vs. LOW exercise trial (P < 0.05). Rates of MyoPS were increased equivalently over 0.5-4.5 h post-exercise recovery (P < 0.05), but remained elevated at 24-28 h post-exercise only following the HIGH trial. In conclusion, an acute bout of high, but not low intensity aerobic exercise in the fasted state resulted in a sustained elevation of both MitoPS and MyoPS at 24-28 h post-exercise recovery.

    March 04, 2014   doi: 10.1152/ajpendo.00487.2013   open full text
  • Brown and White Adipose Tissues. Intrinsic differences in gene expression and response to cold exposure in mice.
    Rosell, M., Kaforou, M., Frontini, A., Okolo, A., Chan, Y.-W., Nikolopoulou, E., Millership, S., Fenech, M. E., MacIntyre, D., Turner, J. O., Moore, J. D., Blackburn, E., Gullick, W. J., Cinti, S., Montana, G., Parker, M. G., Christian, M.
    AJP Endocrinology and Metabolism. February 18, 2014

    Brown adipocytes dissipate energy whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared to white fat, at thermoneutrality, we defined a "BRITE" transcription signature. We identified the genes, pathways and promoter regulatory motifs associated with "browning" as these represent novel targets for understanding this process. For example, Neuregulin 4 was more highly expressed in brown adipose tissue, upregulated in white fat upon cold exposure and cell studies showed it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders including obesity and Type 2 diabetes.

    February 18, 2014   doi: 10.1152/ajpendo.00473.2013   open full text
  • Silencing of the Fibroblast Growth Factor 21 gene is an underlying cause of acinar cell injury in mice lacking MIST1.
    Johnson, C. L., Mehmood, R., Laing, S. W., Stepniak, C. V., Kharitonenkov, A., Pin, C. L.
    AJP Endocrinology and Metabolism. February 18, 2014

    Fibroblast growth factor 21 (FGF21) is a key regulator of metabolism under conditions of stress such as starvation, obesity and hypothermia. Rapid induction of FGF21 is also observed in experimental models of pancreatitis, and FGF21 reduces tissue damage observed in these models, suggesting a non-metabolic function. Pancreatitis is a debilitating disease with significant morbidity that greatly increases the risk of pancreatic ductal adenocarcinoma. The goals of this study were to examine the regulation and function of FGF21 in acinar cell injury, specifically in a mouse model of pancreatic injury (Mist1-/-). Mist1-/- mice exhibit acinar cell disorganization, decreased acinar cell communication and exocytosis, and increased sensitivity to cerulein-induced pancreatitis (CIP). Examination of Fgf21 expression in Mist1-/- mice by qRT-PCR, Northern blot, and Western blot analysis showed a marked decrease in pancreatic Fgf21 expression before and after induction of CIP compared to C57Bl/6 mice. To determine if the loss of FGF21 accounted for the Mist1-/- phenotypes, we generated Mist1-/- mice over-expressing human FGF21 from the ApoE promoter (Mist1-/-ApoE-FGF21). Re-expression of FGF21 partially mitigated pancreatic damage in Mist1-/- tissue based on reduced intrapancreatic enzyme activation, reduced expression of genes involved in fibrosis, and restored cell-cell junctions. Interestingly, alteration of Fgf21 expression in Mist1-/- tissue was not simply due to a loss of direct transcriptional regulation by MIST1. Chromatin immunopreciptation indicated that the loss of Fgf21 in the Mist1-/- pancreas is due, in part, to epigenetic silencing. Thus, our studies identify a new role for FGF21 in reducing acinar cell injury, and uncover a novel mechanism for regulating Fgf21 gene expression.

    February 18, 2014   doi: 10.1152/ajpendo.00559.2013   open full text
  • A systematic survey of lipids across mouse tissues.
    Jain, M., Ngoy, S., Sheth, S. A., Swanson, R. A., Rhee, E. P., Liao, R., Clish, C. B., Mootha, V., Nilsson, R.
    AJP Endocrinology and Metabolism. February 11, 2014

    Lipids are a diverse collection of macromolecules essential for normal physiology, but the tissue distribution and function for many individual lipid species remain unclear. Here, we report a mass spectrometry survey of lipid abundance across 18 mouse tissues, detecting ~1,000 mass spectrometry features, of which we identify 179 lipids from the glycerolipids, glycerophospholipids, lysophospholipids, acylcarnitines, sphingolipids and cholesteryl ester classes. Our data reveals tissue-specific organization of lipids and can be used to generate testable hypotheses. For example, our data indicates that circulating triglycerides positively and negatively associated with future diabetes in humans are enriched in mouse adipose tissue and liver, respectively, raising hypotheses regarding the tissue origins of these diabetes-associated lipids. We also integrate our tissue lipid data with gene expression profiles to predict a number of substrates of lipid-metabolizing enzymes, highlighting choline phosphotransferases and sterol O-acyltransferases. Finally, we identify several tissue-specific lipids not present in plasma under normal conditions that may be of interest as biomarkers of tissue injury, and show that two of these lipids are released into blood following ischemic brain injury in mice. This resource complements existing compendia of tissue gene expression and may be useful for integrative physiology and lipid biology.

    February 11, 2014   doi: 10.1152/ajpendo.00371.2013   open full text
  • Electrical stimulation induces interleukin-6 in skeletal muscle through extracellular ATP, by activating Ca2+ signals and an interleukin-6 autocrine loop.
    Bustamante, M., Fernandez-Verdejo, R., Jaimovich, E., Buvinic, S.
    AJP Endocrinology and Metabolism. February 11, 2014

    Interleukin-6 (IL-6) is an important myokine, highly expressed in skeletal muscle cells upon exercise. We assessed IL-6 expression in response to electrical stimulation (ES) or extracellular ATP, as a known mediator of the excitation-transcription mechanism in skeletal muscle. We examined whether the canonical signaling cascade downstream of IL-6 (IL-6/JAK2/STAT3) also responds to muscle cell excitation concluding that IL-6 influences its own expression through a positive loop. Either ES or exogenous ATP (100 μM) increased both IL-6 expression and pSTAT3 levels in rat myotubes, a process inhibited by 100 μM suramin and 2 U/ml apyrase. ATP also evoked IL-6 expression in both isolated skeletal fibers and extracts derived from whole FDB muscles. ATP increased IL-6 release up to 10 fold. STAT3 activation evoked by ATP was abolished by the JAK2 inhibitor HBC. Blockade of secreted IL-6 with a neutralizing antibody, or pre-incubation with the STAT3 inhibitor VIII, reduced in 70% STAT3 activation evoked by extracellular ATP. Inhibitor VIII also reduced by 70% IL-6 expression evoked by ATP, suggesting a positive IL-6 loop. In addition, ATP increased up to 60% the protein levels of SOCS3, a negative regulator of IL-6 signaling pathway. On the other hand, intracellular calcium chelation or blockade of IP3-dependent calcium signals abolished STAT3 phosphorylation evoked by either extracellular ATP or ES. These results suggest that expression of IL-6 in stimulated skeletal muscle cells is mediated by extracellular ATP and nucleotide receptors, involving IP3-dependent calcium signals as an early step that triggers a positive IL-6 autocrine loop.

    February 11, 2014   doi: 10.1152/ajpendo.00450.2013   open full text
  • Temporal changes in nutritional state affect hypothalamic POMC peptide levels independently of leptin in adult male mice.
    Mercer, A. J., Stuart, R., Attard, C. A., Otero-Corchon, V., Nillni, E. A., Low, M. J.
    AJP Endocrinology and Metabolism. February 11, 2014

    Hypothalamic proopiomelanocortin (POMC) neurons constitute a critical anorexigenic node in the CNS for maintaining energy balance. These neurons directly affect energy expenditure and feeding behavior by releasing bioactive neuropeptides, but are also subject to signals directly related to nutritional state such as the adipokine leptin. To further investigate the interaction of diet and leptin on hypothalamic POMC peptide levels, we exposed 8-10 wk old male POMC-DsRed transgenic reporter mice to either 24-48 h (acute) or 2 wk (chronic) food restriction, high-fat diet (HFD) or leptin treatment. Using semiquantitative immunofluorescence and radioimmunoassays, we discovered that acute fasting and chronic food-restriction decreased the levels of ACTH, α-MSH and β-endorphin in the hypothalamus, together with decreased DsRed fluorescence, compared to control ad libitum fed mice. Furthermore, acute but not chronic HFD or leptin administration selectively increased α-MSH levels in POMC fibers and increased DsRed fluorescence in POMC cell bodies. HFD and leptin treatments comparably increased circulating leptin levels at both time points, suggesting that transcription of Pomc and synthesis of POMC peptide products are not modified in direct relation to the concentration of plasma leptin. Our findings indicate that negative energy balance persistently downregulated POMC peptide levels, and this phenomenon may be partially explained by decreased leptin levels, as these changes were blocked in fasted mice treated with leptin. In contrast, sustained elevation of plasma leptin by HFD or hormone supplementation did not significantly alter POMC peptide levels, indicating that enhanced leptin signaling does not chronically increase Pomc transcription and peptide synthesis.

    February 11, 2014   doi: 10.1152/ajpendo.00540.2013   open full text
  • Hepatic sirtuin 1 is dispensable for fibrate-induced peroxisome proliferator activated receptor {alpha} function in vivo.
    Bonzo, J. A., Brocker, C., Wang, R.-H., Deng, C.-X., Gonzalez, F. J.
    AJP Endocrinology and Metabolism. February 04, 2014

    Peroxisome proliferator activated receptor α (PPARα) mediates metabolic remodeling resulting in enhanced mitochondrial and peroxisomal β-oxidation of fatty acids. In addition to the physiological stimuli of fasting and high fat diet, PPARα is activated by the fibrate class of drugs for the treatment of dyslipidemia. Sirtuin 1 (SIRT1), an important regulator of energy homeostasis, was down-regulated in fibrate treated wild-type mice suggesting PPARα regulation of Sirt1 gene expression. The impact of SIRT1 loss on PPARα functionality in vivo was assessed in hepatocyte-specific knockout mice that lack the deacetylase domain of SIRT1 (Sirt1Liv). Knockout mice were treated with fibrates or fasted for 24 hours to activate PPARα. Basal expression of the PPARα target genes Cyp4a10 and Cyp4a14 was reduced in Sirt1Liv mice compared to wild-type mice. However, no difference was observed between wild-type and Sirt1Liv mice in either fasting- or fibrate-mediated induction of PPARα target genes. Similar to the initial results, there was no difference in fibrate-activated PPARα gene induction. To assess the relationship between SIRT1 and PPARα in a pathophysiological setting, Sirt1Liv mice were maintained on a high fat diet for 14 weeks followed by fibrate treatment. Sirt1Liv mice exhibited increased body mass compared to control mice. In the context of a high fat diet, Sirt1Liv mice did not respond to the cholesterol lowering effects of the fibrate treatment. However, there were no significant differences in PPARα target gene expression. These results suggest that, in vivo, SIRT1 deacetylase activity does not significantly impact induced PPARα activity.

    February 04, 2014   doi: 10.1152/ajpendo.00175.2013   open full text
  • Eukaryotic translation initiation factor 5A inhibition alters physiopathology and immune responses in a "humanized" transgenic mouse model of type 1 diabetes.
    Imam, S., Mirmira, R. G., Jaume, J. C.
    AJP Endocrinology and Metabolism. February 04, 2014

    Therapeutic options for treatment of type 1 diabetes are still missing. New avenues for immune modulation need to be developed. Here we attempted at altering the diabetes outcome of our humanized model of T1D by inhibiting translation-initiation factor eIF5A hypusination in vivo. Double-transgenic (DQ8-GAD65) mice were immunized with adenoviral vectors carrying GAD65 for diabetes induction. Animals were subsequently treated with deoxyhypusine synthase (DHS) inhibitor GC7 and monitored for diabetes development over time. On one hand, helper CD4+ T-cells were clearly affected by the downregulation of the eIF5A not just at the pancreas level but overall. On the other hand, the T regulatory cell component of CD4 responded with activation and proliferation significantly higher than in the non-GC7 treated controls. Female mice seemed to be more susceptible to these effects. All together, our results show for the first time, that downregulation of eIF5A through inhibition of DHS altered the physiopathology and observed immune outcome of diabetes in an animal model that closely resembles human T1D. Although the development of diabetes could not be abrogated by DHS inhibition, the immunomodulatory capacity of this approach may supplement other interventions directed at increasing regulation of autoreactive T cells in T1D.

    February 04, 2014   doi: 10.1152/ajpendo.00537.2013   open full text
  • Impact of embryo number and periconceptional undernutrition on insulin signalling and gluconeogenic factors and microRNAs in the liver of fetal sheep.
    Lie, S., Morrison, J. L., Williams-Wyss, O., Suter, C. M., Humphreys, D. T., Ozanne, S. E., Zhang, S., MacLaughlin, S. M., Kleemann, D. O., Walker, S. K., Roberts, C. T., McMillen, I. C.
    AJP Endocrinology and Metabolism. February 04, 2014

    This study aimed to determine whether exposure of the oocyte and/or embryo to maternal undernutrition results in the later programming of insulin action in the liver and factors regulating gluconeogenesis. To do this, we collect livers from singleton and twin fetal sheep that were exposed to periconceptional (PCUN: -60d-7d) or preimplantation (PIUN: 0d-7d) undernutrition at 136-138d gestation (term=150d). The mRNA and protein abundance of insulin signalling and gluconeogenic factors were then quantified using qRT-PCR and Western blotting, respectively, and global microRNA expression was quantified using deep sequencing methodology. We found that hepatic PEPCK-C mRNA (P<0.01) and protein abundance and the protein abundance of IRS-1 (P<0.01), p110β (P<0.05), PTEN (P<0.05), CREB (P<0.01) and pCREB (Ser133) (P<0.05) were decreased in the PCUN and PIUN singletons. In contrast, hepatic protein abundance of IRS-1 (P<0.01), p85 (P<0.01), p110β (P<0.001), PTEN (P<0.01), Akt2 (P<0.01), pAkt (Ser473) (P<0.01) and pFOXO-1(Thr24) (P<0.01) was increased in twins. There was a decrease in PEPCK-C mRNA (P<0.01), but paradoxically an increase in PEPCK-C protein (P<0.001) in twins. Both PCUN and PIUN altered the hepatic expression of 23 specific microRNAs. We propose that the differential impact of maternal undernutrition in the presence of one or two embryos on mRNAs and proteins involved in the insulin signalling and gluconeogenesis are explained by the changes in the expression of a suite of specific candidate microRNAs.

    February 04, 2014   doi: 10.1152/ajpendo.00553.2013   open full text
  • De-novo synthesis of milk lipids in humans.
    Mohammad, M. A., Sunehag, A. L., Haymond, M. W.
    AJP Endocrinology and Metabolism. February 04, 2014

    Background: Mammary gland(MG) de-novo lipogenesis contributes significantly to milk fat in animals but little is known in humans. Objective: To test the hypothesis that the incorporation of 13C carbons from [U-13C]glucose into fatty acids (FA) and glycerol in triglycerides (TG)will be greater: a)in milk than plasma TG, b)during a high carbohydrate (H-CHO) diet than high fat (H-FAT) diet and c)during feeding than fasting. Materials/Methods: Seven healthy lactating women were studied on two isocaloric, isonitrogenous diets. On one occasion subjects received diets containing H-FAT or H-CHO diet for 1 week. Incorporation of 13C from infused [U-13C]glucose into FA and glycerol was measured using GC/MS and gene expression in RNA isolated from milk fat globule using microarrays. Results: Incorporation of 13C2 into milk FA, increased with increased FA chain length from C2:0 to C12:0 but progressively declined in C14:0 and C16:0 and was not detected in FA>C16. During feeding, regardless of diets, enrichment of 13C2 in milk FA and 13C3 in milk glycerol were ~3 and ~7 fold higher compared to plasma FA and glycerol, respectively. Following an overnight fast during H-CHO and H-FAT diets, 25% and 6%, respectively, of medium chain FA (MCFA, C6-C12) in milk were derived from glucose but increased to 75% and 25% with feeding. Expression of genes involved in FA or glycerol synthesis was unchanged regardless of diet or fast/fed conditions. Conclusions: The human MG is capable of de novo lipogenesis, of primarily MCFA and glycerol, which is influenced by the macronutrient composition of the maternal diet.

    February 04, 2014   doi: 10.1152/ajpendo.00605.2013   open full text
  • Targeted deletion of C1q/TNF-related protein 9 (CTRP9) increases food intake, decreases insulin sensitivity, and promotes hepatic steatosis in mice.
    Wei, Z., Lei, X., Petersen, P. S., Aja, S., Wong, G. W.
    AJP Endocrinology and Metabolism. January 28, 2014

    Transgenic over-expression of CTRP9, a secreted hormone down-regulated in obesity, confers striking protection against diet-induced obesity and type 2 diabetes. The physiological relevance of this adiponectin-related plasma protein, however, remains undefined. Here, we used gene targeting to establish the metabolic function of CTRP9 in a physiological context. Mice lacking CTRP9 were obese and gained significantly more body weight when fed standard laboratory chow. Increased food intake, due in part to up-regulated expression of hypothalamic orexigenic neuropeptides, contributed to greater adiposity in CTRP9 knockout mice. While the frequency of food intake remained unchanged, CTRP9 knockout mice increased caloric intake by increasing meal size and decreasing satiety ratios. The absence of CTRP9 also resulted in peripheral tissue insulin resistance, leading to increased fasting insulin levels, impaired hepatic insulin signaling, and reduced insulin tolerance. Increased expression of lipogenic genes, combined with enhanced caloric intake, contributed to hepatic steatosis in CTRP9 knockout mice. Loss of CTRP9 also resulted in reduced skeletal muscle AMPK activation and mitochondrial content. Together, these results provide the genetic evidence for a physiological role of CTRP9 in controlling energy balance via central and peripheral mechanisms.

    January 28, 2014   doi: 10.1152/ajpendo.00593.2013   open full text
  • Long term niacin treatment induces insulin resistance and adrenergic responsiveness in adipocytes by adaptive down-regulation of phosphodiesterase 3B.
    Heemskerk, M. M., van den Berg, S. A. A., Pronk, A. C. M., Van Klinken, J., Boon, M. R., Havekes, L. M., Rensen, P. C. N., Willems van Dijk, K., van Harmelen, V.
    AJP Endocrinology and Metabolism. January 28, 2014

    The lipid lowering effect of niacin has been attributed to the inhibition of cAMP production in adipocytes, thereby inhibiting intracellular lipolysis and release of non-esterified fatty acids (NEFA) to the circulation. However, long term niacin treatment leads to a normalization of plasma NEFA levels and induces insulin resistance, for which the underlying mechanisms are poorly understood. The current study addressed the effects of long term niacin treatment on insulin-mediated inhibition of adipocyte lipolysis and focused on the regulation of cAMP levels. APOE*3-Leiden.CETP transgenic mice treated with niacin for 15 weeks were subjected to an insulin tolerance test and showed whole body insulin resistance. Similarly, adipocytes isolated from niacin treated mice were insulin resistant and, interestingly, exhibited an increased response to cAMP stimulation by 8Br-cAMP, β1 and β2-adrenergic stimulation. Gene expression analysis of the insulin and β-adrenergic pathways in adipose tissue indicated that all genes were down-regulated, including the gene encoding the cAMP degrading enzyme phosphodiesterase 3B (PDE3B). In line with this, we showed that insulin induced a lower PDE3B response in adipocytes isolated from niacin treated mice. Inhibiting PDE3B with cilostazol increased lipolytic responsiveness to cAMP stimulation in adipocytes. These data show that long term niacin treatment leads to a down-regulation of PDE3B in adipocytes which could explain part of the observed insulin resistance and the increased responsiveness to cAMP stimulation.

    January 28, 2014   doi: 10.1152/ajpendo.00641.2013   open full text
  • Partial ablation of leptin signaling in mouse pancreatic {alpha}-cells does not alter either glucose or lipid homeostasis.
    Tuduri, E., Denroche, H. C., Kara, J. A., Asadi, A., Fox, J. K., Kieffer, T. J.
    AJP Endocrinology and Metabolism. January 28, 2014

    The role of glucagon in the pathological condition of diabetes is gaining interest, and it has been recently reported that its action is essential for hyperglycemia to occur. Glucagon levels, which are elevated in some diabetic models, are reduced following leptin therapy. Likewise, hyperglycemia is corrected in type 1 diabetic mice treated with leptin, although the mechanisms have not been fully determined. A direct inhibitory effect of leptin on mouse and human α-cells has been demonstrated at the levels of electrical activity, calcium signaling and glucagon secretion. In the present study we employed the Cre-loxP strategy to generate Leprflox/flox Gcg-cre mice, which specifically lack leptin receptors in glucagon-secreting α-cells, in order to determine whether leptin resistance in α-cells contributes to hyperglucagonemia, and also whether leptin action in α-cells is required to improve glycemia in type 1 diabetes with leptin therapy. Immunohistochemical analysis of pancreas sections revealed Cre-mediated recombination in ~43% of the α-cells. We observed that in vivo Leprflox/flox Gcg-cre mice display normal glucose and lipid homeostasis. In addition, leptin administration in streptozotocin (STZ)-induced diabetic Leprflox/flox Gcg-cre mice restored euglycemia similarly to control mice. These findings suggest that loss of leptin receptor signaling in close to half of α-cells does not alter glucose metabolism in vivo, nor is it sufficient to prevent the therapeutic action of leptin in type 1 diabetes.

    January 28, 2014   doi: 10.1152/ajpendo.00681.2013   open full text
  • Overnutrition induces {beta}-cell differentiation through prolonged activation of {beta} cells in zebrafish larvae.
    Li, M., Maddison, L. A., Page-McCaw, P., Chen, W.
    AJP Endocrinology and Metabolism. January 28, 2014

    Insulin from islet β cells maintains glucose homeostasis by stimulating peripheral tissues to remove glucose from circulation. Persistent elevation of insulin demand increases β-cell number through self-replication or neogenesis (differentiation) as part of a compensatory response. However, it is not well understood how a persistent increase of insulin demand is detected. We have previously demonstrated that a persistent increase of insulin demand by overnutrition induces compensatory β-cell differentiation in zebrafish. Here, we use a series of pharmacological and genetic analyses to show that prolonged stimulation of existing β cells is necessary and sufficient for the compensatory response. In the absence of feeding, tonic, but not intermittent, pharmacological activation of β-cell secretion was sufficient to induce β-cell differentiation. Conversely, drugs that block β-cell secretion, including a KATP channel agonist and an L-type Ca2+ channel blocker, suppressed the overnutrition response. Genetic experiments specifically targeting β cells confirm existing β cells as the overnutrition sensor. First, inducible expression of a constitutively active KATP channel in β cells suppressed the overnutrition effect. Second, inducible expression of a dominant-negative KATP mutant induced β-cell differentiation independent of nutrients. Third, sensitizing β-cell metabolism by transgenic expression of a hyperactive GCK potentiated differentiation. Finally, ablation of the existing β cells abolished the differentiation response. Taken together, these data establish that overnutrition induces β-cell differentiation in larval zebrafish through prolonged activation of β cells. These findings demonstrate an essential role for existing β cells in sensing overnutrition and compensating for their own insufficiency by recruiting additional β cells.

    January 28, 2014   doi: 10.1152/ajpendo.00686.2013   open full text
  • Estradiol modulates Kiss1 Neuronal Response to Ghrelin.
    Frazao, R., Dungan-Lemko, H., Silva, R. P. d., ratra, D. V., Lee, C. E., Williams, K. W., Zigman, J. M., Elias, C. F.
    AJP Endocrinology and Metabolism. January 28, 2014

    Ghrelin is a metabolic signal regulating energy homeostasis. Circulating ghrelin levels rise during starvation and fall after a meal and, therefore, ghrelin may function as a signal of negative energy balance. Ghrelin may also act as a modulator of reproductive physiology, as acute ghrelin administration suppresses gonadotropin secretion and inhibits the neuroendocrine reproductive axis. Interestingly, ghrelin's effect in female metabolism varies according to the estrogen milieu predicting an interaction between ghrelin and estrogens, likely at the hypothalamic level. Here we show that ghrelin receptor (GHSR) and estrogen receptor α (ERα) are coexpressed in several hypothalamic sites. Higher levels of circulating estradiol increased the expression of GHSR mRNA and the co-expression of GHSR mRNA and ERα selectively in the arcuate nucleus (Arc). Subsets of preoptic and Arc Kiss1 neurons coexpressed GHSR. Increased colocalization was observed in Arc Kiss1 neurons of ovariectomized estradiol-treated (OVX+E2, 80%) compared to ovariectomized oil-treated (OVX, 25%) mice. Acute actions of ghrelin on Arc Kiss1 neurons were also modulated by estradiol; 75% and 22% of Kiss1 neurons of OVX+E2 and OVX mice respectively depolarized in response to ghrelin. Our findings indicate that ghrelin and estradiol may interact in several hypothalamic sites. In the Arc, high levels of E2 increase GHSR mRNA expression, modifying the colocalization rate with ERα and Kiss1 and the proportion of Kiss1 neurons acutely responding to ghrelin. Our findings indicate that E2 alters the responsiveness of Kisspeptin neurons to metabolic signals, potentially acting as a critical player in the metabolic control of the reproductive physiology.

    January 28, 2014   doi: 10.1152/ajpendo.00211.2013   open full text
  • Sphingosine Kinase 1 Regulates Adipose Proinflammatory Responses and Insulin Resistance.
    Wang, J., Badeanlou, L., Bielawski, J., Ciaraldi, T. P., Samad, F.
    AJP Endocrinology and Metabolism. January 28, 2014

    Adipose dysfunction resulting from chronic inflammation and impaired adipogenesis is increasingly recognized as a major contributor to obesity-mediated insulin resistance, but the molecular mechanisms that maintain healthy adipocytes and limit adipose inflammation remain unclear. Here, we used genetic and pharmacological approaches to delineate a novel role for sphingosine kinase 1 (SK1) in metabolic disorders associated with obesity. SK1 phosphorylates sphingosine to form sphingosine 1 phosphate (S1P), a bioactive sphingolipid with numerous roles in inflammation. SK1 mRNA expression was increased in adipose tissue of diet-induced obese (DIO) mice and obese type 2 diabetic humans. In DIO mice, SK1 deficiency increased markers of adipogenesis and adipose gene expression of the anti-inflammatory molecules IL-10 and adiponectin, and reduced adipose tissue macrophage (ATM) recruitment and proinflammatory molecules TNF-α and IL-6. These changes were associated with enhanced insulin signaling in adipose and muscle and improved systemic insulin sensitivity and glucose tolerance in SK1-/- mice. Specific pharmacological inhibition of SK1 in WT DIO mice also reduced adipocyte and ATM inflammation and improved overall glucose homeostasis. These data suggest that the SK1/S1P axis could be an attractive target for the development of treatments to ameliorate adipose inflammation and insulin resistance associated with obesity and T2D.

    January 28, 2014   doi: 10.1152/ajpendo.00549.2013   open full text
  • The effect of IL-6 on the insulin sensitivity in patients with type 2 diabetes.
    Harder-Lauridsen, N. M., Krogh-Madsen, R., Holst, J. J., Plomgaard, P., Leick, L., Pedersen, B. K., Fischer, C. P.
    AJP Endocrinology and Metabolism. January 28, 2014

    Elevated interleukin-6 (IL-6) levels are associated with type 2 diabetes, but its role in glucose metabolism is controversial. We investigated the effect of IL-6 on insulin-stimulated glucose metabolism in type 2 diabetes patients, and hypothesized that an acute, moderate IL-6 elevation would increase the insulin-mediated glucose uptake. Men with type 2 diabetes not treated with insulin (n=9, age [mean±SD] 54.9±9.7 years, BMI 34.8±6.1 kg/m2, HbA1c % 7.0±1.0) received continuous intravenous infusion with either recombinant human IL-6 (rhIL-6) or placebo. After one hour with placebo or rhIL-6, a 3-hour hyperinsulinemic-isoglycemic clamp was initiated. Whole body glucose metabolism was measured using stable isotope labeled tracers. Signal transducer and activator of transcription 3 (STAT3) phosphorylation and suppressor of cytokine signaling 3 (SOCS3) expression were measured in muscle biopsies. Whole body energy expenditure was measured using indirect calorimetry. In response to the infusion of rhIL-6, circulating levels of IL-6 (P<0.001), neutrophils (P<0.001), and cortisol (P<0.001) increased, while lymphocytes decreased (P<0.01). However, IL-6 infusion did not change glucose infusion rate, rate of appearance, or rate of disappearance during the clamp. While IL-6 enhanced phosphorylation of STAT3 in skeletal muscle (P=0.041), the expression of SOCS3 remained unchanged. Whole body oxygen uptake (P<0.01) and expired carbon dioxide (P<0.01) increased during rhIL-6 infusion. In summary, although IL-6 induced local and systemic responses, the insulin-stimulated glucose uptake was not affected. While different contributing factors may be involved, our results are in contrast to our hypothesis as well as previous findings in young, healthy men.

    January 28, 2014   doi: 10.1152/ajpendo.00571.2013   open full text
  • Local Overexpression of the Myostatin Propeptide Increases Glucose Transporter Expression and Enhances Skeletal Muscle Glucose Disposal.
    Cleasby, M. E., Jarmin, S., Eilers, W., Elashry, M., Andersen, D. K., Dickson, G., Foster, K.
    AJP Endocrinology and Metabolism. January 28, 2014

    Insulin resistance (IR) in skeletal muscle is a pre-requisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopaenic obesity. The molecular defects that underpin this syndrome are not well characterised and there is no licenced treatment. Deletion of the transforming growth factor-β family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/ latency associated peptide (ProMyo), results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo into right tibialis cranialis/ extensor digitorum longus muscles of rats and saline into left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7% and 11%) and showed increased radiolabelled 2-deoxyglucose uptake (26% and 47%) and glycogen storage (28% and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signalling intermediates and AMP-activated kinase were slightly decreased, possibly due to reduced expression of insulin-like growth factor-1 in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone and this approach may offer potential for the therapy of IR syndromes.

    January 28, 2014   doi: 10.1152/ajpendo.00586.2013   open full text
  • Insulinotropic Effect of High Potassium Concentration beyond Plasma Membrane Depolarization.
    Belz, M., Willenborg, M., Gorgler, N., Hamada, A., Schumacher, K., Rustenbeck, I.
    AJP Endocrinology and Metabolism. January 22, 2014

    The question whether K+ depolarization is an appropriate experimental substitute for the physiological nutrient-induced depolarization of the beta cell plasma membrane was investigated using primary mouse beta cells and islets. At basal glucose 40 mM K+ induced a massive monophasic response, whereas 15 mM K+ had only a minimal insulinotropic effect, even though the increase in the cytosolic Ca2+ concentration ([Ca2+]i) was not inferior to that by 20 mM glucose. In voltage-clamp experiments Ca2+ influx appeared as nifedipine-inhibitable inward action currents in the presence of sulphonylurea plus TEA to block compensatory outward K+ currents. Under these conditions 15 mM K+ induced prolonged action currents and 40 mM K+ transformed the action current pattern into a continuous inward current. Correspondingly, 15 mM K+ led to an oscillatory increase, 40 mM K+ to a plateau of [Ca2+]i superimposed on the [Ca2+]i elevated by sulfonylurea plus TEA. Raising K+ to 15 or 40 mM in the presence of sulfonylurea (+/- TEA) led to a fast further increase of insulin secretion. This was reduced to basal levels by nifedipine or CoCl2. The effects of 15 mM K+ on depolarization, action currents and insulin secretion were mimicked by adding 35 mM Cs+, those of 40 mM K+ by adding 35 mM Rb+, in parallel with their ability to substitute for K+ as permeant cation. In conclusion, the alkali metals, K+, Rb+ or Cs+ concentration-dependently transform the pattern of Ca2+ influx into the beta cell and may thus generate stimuli of supraphysiological strength for insulin secretion.

    January 22, 2014   doi: 10.1152/ajpendo.00362.2013   open full text
  • Regulation of body temperature and brown adipose tissue thermogenesis by bombesin receptor subtype-3.
    Lateef, D. M., Abreu-Vieira, G., Xiao, C., Reitman, M. L.
    AJP Endocrinology and Metabolism. January 22, 2014

    Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, with Brs3 knockout (Brs3-/y) mice being hypometabolic, hypothermic, and hyperphagic and developing obesity. We now report that the reduced body temperature is more readily detected if body temperature is analyzed as a function of physical activity level and light/dark phase. Physical activity level correlated best with body temperature four minutes later. The Brs3-/y metabolic phenotype is not due to intrinsically impaired brown adipose tissue function or in the communication of sympathetic signals from the brain to brown adipose tissue, since Brs3-/y mice have intact thermogenic responses to stress, acute cold exposure, and β3 adrenergic activation, and Brs3-/y mice prefer a cooler environment. Treatment with the BRS-3 agonist MK-5046 increased brown adipose tissue temperature and body temperature in wild type, but not Brs3-/y mice. Intrahypothalamic infusion of MK-5046 increased body temperature. These data indicate that the BRS-3 regulation of body temperature is via a central mechanism, upstream of sympathetic efferents. The reduced body temperature in Brs3-/y mice is due to altered regulation of energy homeostasis affecting higher center regulation of body temperature, rather than an intrinsic defect in brown adipose tissue.

    January 22, 2014   doi: 10.1152/ajpendo.00615.2013   open full text
  • The biochemistry and cell biology of aging: metabolic regulation through mitochondrial signaling.
    Long, Y. C., Tan, T. M. C., Inoue, T., Tang, B. L.
    AJP Endocrinology and Metabolism. January 22, 2014

    Cellular and organ metabolism affects organismal lifespan. Aging is characterized by increased risks for metabolic disorders, with age-associated degenerative diseases exhibiting varying degrees of mitochondrial dysfunction. The traditional view of the role of mitochondria generated reactive oxygen species (ROS) in cellular aging, assumed to be causative and simply detrimental for a long time now, is in need of reassessment. While there is little doubt that high levels of ROS are detrimental, mounting evidence points towards a lifespan extension effect exerted by mild to moderate ROS elevation. Dietary caloric restriction (CR), inhibition of insulin-like growth factor (IGF)-1 signaling, and inhibition of the nutrient-sensing mechanistic target of rapamycin (mTOR) are robust longevity promoting interventions. All of these appear to elicit mitochondrial retrograde signaling processes (defined as signaling from the mitochondria to the rest of the cell, for example, the mitochondrial unfolded protein response, or UPRmt). The effects of mitochondrial retrograde signaling may even spread to other cells/tissues in a non-cell autonomous manner by yet unidentified signaling mediators. Multiple recent publications support the notion that an evolutionarily conserved, mitochondria-initiated signaling is central to the genetic and epigenetic regulation of cellular aging and organismal lifespan.

    January 22, 2014   doi: 10.1152/ajpendo.00665.2013   open full text
  • The effects of insulin resistance on skeletal muscle growth and exercise capacity in type 2 diabetic mouse models.
    Ostler, J. E., Maurya, S. K., Dials, J., Roof, S. R., Devor, S. T., Ziolo, M. T., Periasamy, M.
    AJP Endocrinology and Metabolism. January 14, 2014

    Type 2 diabetes mellitus is associated with an accelerated muscle loss during aging, decreased muscle function, and increased disability. To better understand the mechanisms causing this muscle deterioration in type 2 diabetes, we assessed muscle weight, exercise capacity, and biochemistry in db/db and TallyHo mice at prediabetic and overtly diabetic ages. Maximum running speeds and muscle weights were already reduced in prediabetic db/db mice when compared to lean controls and more severely reduced in the overtly diabetic db/db mice. In contrast to db/db mice, TallyHo muscle size dramatically increased and maximum running speed was maintained during the progression from prediabetes to overt diabetes. Analysis of mechanisms that may contribute to decreased muscle weight in db/db mice demonstrated that insulin-dependent phosphorylation of enzymes that promote protein synthesis was severely blunted in db/db muscle. In addition, prediabetic (6 week old) and diabetic (12 week old) db/db muscle exhibited an increase in a marker of proteasomal protein degradation - the level of polyubiquitinated proteins. Chronic treadmill training of db/db mice improved glucose tolerance, exercise capacity, and reduced markers of protein degradation, but only mildly increased muscle weight. The differences in muscle phenotype between these models of type 2 diabetes suggest that insulin resistance and chronic hyperglycemia alone are insufficient to rapidly decrease muscle size and function and that the effects of diabetes on muscle growth and function are animal model-dependent.

    January 14, 2014   doi: 10.1152/ajpendo.00277.2013   open full text
  • Muscle RING Finger-1 Attenuates IGF-1-dependent cardiomyocyte hypertrophy by Inhibiting JNK Signaling.
    Wadosky, K. M., Rodriguez, J. E., Hite, R. L., Min, J.-N., Walton, B., Willis, M. S.
    AJP Endocrinology and Metabolism. January 14, 2014

    Recent studies implicate the muscle-specific ubiquitin ligase muscle RING finger-1 (MuRF1) in inhibiting pathological cardiomyocyte growth in vivo by inhibiting the transcription factor SRF. These studies led us to hypothesize that MuRF1 similarly inhibits insulin-like growth factor-1 (IGF-1)-mediated physiologic cardiomyocyte growth. We identified two lines of evidence to support this hypothesis: 1) IGF-1 stimulation of cardiac-derived cells with MuRF1 knockdown exhibited an exaggerated hypertrophy; 2) Conversely, increased MuRF1 expression abolished IGF-1-dependent cardiomyocyte growth. Enhanced hypertrophy with MuRF1 knockdown was accompanied by increases in Akt-regulated gene expression. Unexpectedly, MuRF1 inhibition of this gene expression profile was not a result of differences in p-Akt). Instead, we found MuRF1 inhibits total protein levels of Akt, GSK3β (downstream of Akt) and mTOR, while limiting c-Jun protein expression, a mechanism recently shown to govern Akt, GSK3β, and mTOR activities and expression. These findings establish that MuRF1 inhibits IGF-1 signaling by restricting c-Jun activity, a novel mechanism recently identified in the context of ischemia reperfusion injury. Since IGF-1 regulates exercise-mediated physiological cardiac growth, we challenged MuRF1 -/- and MuRF1Tg+ mice and their wildtype sibling controls to 5 weeks of voluntary wheel running. MuRF1 -/- cardiac growth was significantly increased over wildtype control; converse the enhanced exercise-induced cardiac growth was lost in MuRF1Tg+ animals. These studies demonstrate that MuRF1-dependent attenuation of IGF-1 signaling via c-Jun is applicable in vivo and establish that further understanding of this novel mechanism may be crucial in the development of therapies targeting IGF-1 signaling.

    January 14, 2014   doi: 10.1152/ajpendo.00326.2013   open full text
  • Disturbed intestinal nitrogen homeostasis in a mouse model of high-fat diet-induced obesity and glucose intolerance.
    Do, T. T. H., Hindlet, P., Waligora-Dupriet, A.-J., Kapel, N., Neveux, N., Mignon, V., Delomenie, C., Farinotti, R., Feve, B., Buyse, M.
    AJP Endocrinology and Metabolism. January 14, 2014

    The oligopeptide transporter PEPT1 plays a major role in the regulation of nitrogen supply, since it is responsible for 70% of the dietary nitrogen absorption. Previous studies demonstrated that PEPT1 expression and function in jejunum are reduced in diabetes and obesity, suggesting a nitrogen malabsorption from the diet. Surprisingly, we reported here a decrease in gut nitrogen excretion in high-fat diet (HFD)-fed mice and further investigated the mechanisms that could explain this apparent contradiction.. Upon HFD, mice exhibited an increased concentration of free amino acids (AAs) in the portal vein (60%) along with a selective increase in the expression of two AA transporters (Slc6a20a, Slc36a1), pointing to a specific and adaptive absorption of some AAs. A delayed transit time (+ 40%) and an increased intestinal permeability (+ 80%) also contribute to the increase in nitrogen absorption. Besides, HFD mice exhibited a 2.2-fold decrease in fecal DNA resulting from a reduction in nitrogen catabolism from cell desquamation and/or in the intestinal microbiota. Indeed, major quantitative (2.5-fold reduction) and qualitative alterations of intestinal microbiota were observed in feces of HFD mice. Collectively, our results strongly suggest that both increased AA transporters, intestinal permeability and transit time, and changes in gut microbiota are involved in the increased circulating AA levels. Modifications in nitrogen homestasis provides a new insight in HFD-induced obesity and glucose intolerance, however whether these modifications are beneficial or detrimental for the HFD-associated metabolic complications remain an open issue.

    January 14, 2014   doi: 10.1152/ajpendo.00437.2013   open full text
  • Interleukin-1{beta} hampers glucose stimulated insulin secretion in Cohen diabetic rat islets through mitochondrial cytochrome c oxidase inhibition by nitric-oxide.
    Weksler-Zangen, S., Aharon-Hananel, G., Mantzur, C., Aouizera, T., Gurgul-Convey, E., Raz, I., Saada, A.
    AJP Endocrinology and Metabolism. January 14, 2014

    Aims/hypothesis High-sucrose-low-copper-diet (HSD) induces inhibition of glucose-stimulated-insulin-secretion (GSIS), pancreatic-acinar-cell apoptosis and infiltration of interleukin-1β expressing-macrophages in hyperglycemic-Cohen-diabetes-sensitive rats (CDs), but not in Cohen-diabetes-resistant rats (CDr). Copper-supplemented-HSD increased activity of the copper-dependent mitochondrial-respiratory-chain-enzyme, cytochrome-c-oxidase (COX) and reversed hyperglycemia. This study examined the mechanism by which interleukin-1β modulates GSIS and the role of COX in this process. Methods We measured COX-activity, ATP-content, GSIS, iNOS expression and nitrite-production in isolated-islets of CDs and CDr fed different-diets, +/- interleukin-1β and N-nitro-L-arginine, copper or potassium-cyanide. Results A parallel reduction in COX-activity, ATP-content and GSIS was exhibited by isolated-islets of CDs-rats, fed regular-diet. These were severely-reduced following HSD and were restored to regular-diet levels on copper-supplemented-HSD (p<0.01 vs. CDr-islets). Potassium-cyanide chemically reduced COX-activity, decreasing GSIS, thus, reinforcing the link between islet-COX-activity and GSIS. Interleukin-1β (2.5U/ml) reduced GSIS and COX-activity in CDs-islets. Exposure to 10U/ml interleukin-1β decreased GSIS and COX-activity in both CDs and CDr islets inducing a similar nitrite production. Nevertheless, the effect on GSIS was more marked in CDs-islets. A significant iNOS expression was detected in CDs on HSD diet, which was reduced by copper-supplementation. N-nitro-L-arginine, and copper prevented the deleterious-effect of interleukin-1β on COX-activity and GSIS. Conclusions/interpretation Reduced islets-COX-activity renders vulnerability to GSIS-inhibition on low-copper-HSD through two interrelated pathways, 1) by further reducing the activity of COX essential for β-cell ATP-production and insulin-secretion, and, 2) by inducing the expression of iNOS and nitric-oxide-mediated COX-inhibition. We suggest, that islet COX-activity must be maintained above a critical-threshold to sustain adequate-GSIS with exposure to low-copper-HSD.

    January 14, 2014   doi: 10.1152/ajpendo.00451.2013   open full text
  • Activation of alternative NF-{kappa}B signaling during recovery of disuse-induced loss of muscle oxidative phenotype.
    Remels, A. H., Pansters, N. A. M., Gosker, H. R., Schols, A. M. W. J., Langen, R. C. J.
    AJP Endocrinology and Metabolism. January 14, 2014

    BACKGROUND: Physical inactivity-induced loss of skeletal muscle oxidative phenotype (OXPHEN), often observed in chronic disease, adversely affects physical functioning and quality of life. Potential therapeutic targets remain to be identified as the molecular mechanisms involved in reloading-induced recovery of muscle OXPHEN remain incompletely understood. We hypothesized a role for alternative NF-B, as a recently identified positive regulator of muscle OXPHEN, in reloading-induced alterations in muscle OXPHEN. METHODS: Markers and regulators (including alternative NF-B signaling) of muscle OXPHEN were investigated in gastrocnemius muscle of mice subjected to a hind limb suspension/reloading (HLS/RL) protocol. RESULTS: Expression levels of oxidative phosphorylation (OXPHOS) sub-units and slow myosin heavy chain (MyHC) isoforms I and IIA increased rapidly upon RL. After an initial decrease upon HLS, mRNA levels of peroxisome proliferator-activated receptor (PPAR) gamma co-activator (PGC) molecules PGC-1α and PGC-1β and mRNA levels of mitochondrial transcription factor A (Tfam) and estrogen-related receptor α (ERR-α) increased upon RL. PPAR-, nuclear respiratory factor 1 (NRF-1), NRF-2α and sirtuin 1 mRNA levels increased during RL although expression levels were unaltered upon HLS. In addition, both Tfam and NRF-1 protein levels increased significantly during the RL period. Moreover, upon RL, IKK-α mRNA and protein levels increased and phosphorylation of P100 and subsequent processing to P52 was elevated, reflecting alternative NF-B activation. CONCLUSION: RL-induced recovery of muscle OXPHEN is associated with activation of alternative NF-B signaling.

    January 14, 2014   doi: 10.1152/ajpendo.00452.2013   open full text
  • Early mitochondrial dysfunction in glycolytic muscle, but not oxidative muscle, of the fructose-fed insulin-resistant rat.
    Warren, B. E., Lou, P.-H., Lucchinetti, E., Zhang, L., Clanachan, A. S., Affolter, A., Hersberger, M., Zaugg, M., Lemieux, H.
    AJP Endocrinology and Metabolism. January 14, 2014

    Although evidence has been accumulating that type 2 diabetes mellitus (T2DM) is accompanied by mitochondrial dysfunction in skeletal muscle, a causal link between mitochondrial dysfunction and the pathogenesis of the disease remains unclear. Our study focuses on an early stage of the disease in order to determine whether mitochondrial dysfunction contributes to the development of T2DM. The fructose-fed (FF) rat was used as an animal model of early T2DM. Mitochondrial respiration and acylcarnitine species were measured in oxidative (soleus) and glycolytic (extensor digitorum longus, EDL) muscle. Although FF rats displayed characteristic signs of T2DM, including hyperglycemia, hyperinsulinemia, hypertriglyceridemia, mitochondrial content was preserved in both muscles from FF rats. The EDL muscle had reduced complex I and complex I+II respiration in the presence of pyruvate, but not glutamate. The decrease in pyruvate-supported respiration was due to a decrease in pyruvate dehydrogenase activity. Accumulation of C14:1 and C14:2 acylcarnitine species and a decrease in respiration supported by long-chain acylcarnitines but not acetylcarnitine indicated dysfunctional β-oxidation in the EDL muscle. In contrast, the soleus muscle showed preserved mitochondrial respiration, pyruvate dehydrogenase activity and increased fatty acid oxidation as evidenced by overall reduced acylcarnitine levels. Aconitase activity, a sensitive index of reactive oxygen species production in mitochondria, was exclusively reduced in EDL muscle, which showed lower levels of the antioxidant enzymes thioredoxin reductase and glutathione peroxidase. We here show that the glycolytic EDL muscle is more prone to an imbalance between energy supply and oxidation caused by insulin resistance than the oxidative soleus muscle.

    January 14, 2014   doi: 10.1152/ajpendo.00511.2013   open full text
  • 3D islet graft histology: panoramic imaging of neural plasticity in sympathetic reinnervation of transplanted islets under the kidney capsule.
    Juang, J.-H., Peng, S.-J., Kuo, C.-H., Tang, S.-C.
    AJP Endocrinology and Metabolism. January 14, 2014

    Microscopic examination of transplanted islets in an ectopic environment provides information to evaluate islet engraftment, including revascularization and reinnervation. However, due to the dispersed nature of blood vessels and nerves, global visualization of the graft neurovascular network has been difficult. In this research we revealed the neurovascular network by preparing transparent mouse islet grafts under the kidney capsule with optical clearing to investigate the sympathetic reinnervation via 3-dimensional (3D) confocal microscopy. Normoglycemic and streptozotocin-induced diabetic mice were used in syngeneic islet transplantation, with both groups maintaining euglycemia after transplantation. Triple staining of insulin/glucagon, blood vessels, and tyrosine hydroxylase (sympathetic marker) was used to reveal the graft microstructure, vasculature, and sympathetic innervation. Three weeks after transplantation we observed peri-graft sympathetic innervation similar to the peri-islet sympathetic innervation in the pancreas. Six weeks after transplantation prominent intra-graft, perivascular sympathetic innervation was achieved, resembling the pancreatic intra-islet, perivascular sympathetic innervation in situ. Meanwhile, in diabetic recipients, a higher graft sympathetic nerve density was found compared with grafts in normoglycemic recipients, indicating the graft neural plasticity in response to the physiological difference of the recipients and the resolving power of this imaging approach. Overall, this new graft imaging method provides a useful tool to identify the islet neurovascular complex in an ectopic environment to study islet engraftment.

    January 14, 2014   doi: 10.1152/ajpendo.00515.2013   open full text
  • Minimal modeling of insulin secretion in the perfused rat pancreas: a drug effect case study.
    Riz, M., Pedersen, M. G., Toffolo, G. M., Haschke, G., Schneider, H.-C., Klabunde, T., Margerie, D., Cobelli, C.
    AJP Endocrinology and Metabolism. January 14, 2014

    The experimental protocol of the perfused rat pancreas is commonly used to evaluate beta-cell function. In this context mathematical models become useful tools through the determination of indexes that allow the assessment of beta-cell function in different experimental groups, and the quantification of the effects of anti-diabetic drugs, secretagogues, or treatments. However, a minimal model applicable to the isolated perfused rat pancreas was unavailable so far. In this work, we adapt the C-peptide minimal model, previously applied to the intravenous glucose tolerance test, to obtain a specific model for the experimental settings of the perfused pancreas. Using the model, it is possible to estimate indexes describing beta-cell responsivity for first (D) and second phase (S, T) of insulin secretion. The model was initially applied to untreated pancreata, and afterwards used for the assessment of pharmacologically relevant agents (the gut hormone GLP1, the potent GLP1-receptor agonist, lixisenatide, and a GPR40/FFAR1 agonist, SAR1), to quantify and differentiate their effect on insulin secretion. Model fit was satisfactory and parameters were estimated with good precision for both untreated and treated pancreata. Model application showed that lixisenatide reaches improvement of beta-cell function similar to GLP1 (11.7 vs 13.1 fold increase in D and 2.3 vs 2.8 fold increase in S) and demonstrated that SAR1 leads to an additional improvement of beta-cell function even in the presence of postprandial GLP1 levels.

    January 14, 2014   doi: 10.1152/ajpendo.00603.2013   open full text
  • Enhanced activation of cellular AMPK by dual small molecule treatment: AICAR and A769662.
    Ducommun, S., Ford, R. J., Bultot, L., Deak, M., Bertrand, L., Kemp, B. E., Steinberg, G. R., Sakamoto, K.
    AJP Endocrinology and Metabolism. January 14, 2014

    The AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Activation of AMPK provides beneficial outcomes fighting against metabolic disorders such as insulin resistance and type 2 diabetes. Currently, there is no allosteric AMPK activator available for the treatment of metabolic diseases and limited compounds available to robustly stimulate cellular/tissue AMPK in a specific manner. Here, we investigated if simultaneous administration of two different pharmacological AMPK activators, which bind and act on different sites, would result in an additive or synergistic effect on AMPK and its downstream signaling and physiological events in intact cells. We observed that co-treating primary hepatocytes with an AMP mimetic AICAR and a low dose (1 μM) of an allosteric activator A769662 produces a synergistic effect on AMPK Thr172 phosphorylation and catalytic activity, which was associated with a more profound increase/decrease in phosphorylation of downstream AMPK targets and inhibition of hepatic lipogenesis compared to single compound treatment. Mechanistically, we found that co-treatment does not stimulate LKB1, upstream kinase for AMPK, but it protects against dephosphorylation of Thr172 phosphorylation by protein phosphatase, PP2Cα, in an additive manner in cell-free assay. Collectively, we demonstrate that AICAR sensitizes the effect of A769662 and promote AMPK activity and its downstream events. The study demonstrates the feasibility of promoting AMPK activity by using two activators with distinct modes of action in order to achieve a greater activation of AMPK and downstream signaling.

    January 14, 2014   doi: 10.1152/ajpendo.00672.2013   open full text
  • The Identification of Irisin in Human Cerebrospinal Fluid: Influence of Adiposity, Metabolic Markers and Gestational Diabetes.
    Piya, M. K., Harte, A. L., Sivakumar, K., Tripathi, G., Voyias, P. D., James, S., Sabico, S., Al -Daghri, N. M., Saravanan, P., Barber, T. M., Kumar, S., Vatish, M., McTernan, P. G.
    AJP Endocrinology and Metabolism. January 07, 2014

    Background: Peripheral action of irisin improves glucose homeostasis and increases energy expenditure, with no data on a central role of irisin in metabolism. These studies sought to examine (1) presence of irisin in human cerebrospinal fluid (CSF) and banked human hypothalamic tissue, (2) serum irisin in maternal subjects across varying adiposities with or without gestational diabetes (GDM), and (3) their respective neonate offspring. Methods: CSF, serum and neonatal cord serum were collected from 91 pregnant women with and without GDM attending for an elective Caesarean section (BMI: 37.7±7.6 Kg/m2; age: 32±8.3 years). Irisin was assessed by ELISA and correlated with biochemical and anthropometric data. Irisin expression was examined in human hypothalamus by immunohistochemical staining. Results: Serum irisin in pregnant women was significantly lower in non-obese compared to obese and GDM subjects, after adjusting for BMI, lipids and glucose. Irisin was present in neonatal cord serum (237±8ng/ml) and maternal CSF (32±1.5ng/ml). CSF irisin correlated positively with serum irisin levels from non-obese and obese pregnant women (p<0.01), with CSF irisin significantly raised in GDM subjects (p<0.05). Irisin was present in human hypothalamic sections in the paraventricular neurons, co-localized with neuropeptideY. Conclusions: Irisin was detectable in CSF and in paraventricular neurons. Maternal serum irisin was lower in non-obese pregnant women after adjusting for BMI and a number of metabolic parameters. These studies indicate that irisin may have a central role in metabolism in addition to the known peripheral role. Further studies investigating the central action of irisin in human metabolic disease are required.

    January 07, 2014   doi: 10.1152/ajpendo.00308.2013   open full text
  • Long-term ketogenic diet causes glucose intolerance and reduced beta and alpha cell mass but no weight loss in mice.
    Ellenbroek, J. H., van Dijck, L., Tons, H. A., Rabelink, T. J., Carlotti, F., Ballieux, B. E., de Koning, E. J.
    AJP Endocrinology and Metabolism. January 07, 2014

    High-fat, low-carbohydrate ketogenic diets (KD) are used for weight loss and for treatment of refractory epilepsy. Recently, short-time studies in rodents have shown that besides their beneficial effect on body weight, KD lead to glucose intolerance and insulin resistance. However, the long-term effects on pancreatic endocrine cells are unknown. In this study we investigate the effects of long-term KD on glucose tolerance and beta and alpha cell mass in mice. Despite an initial weight loss, KD did not result in weight loss after 22 weeks. Plasma markers associated with dyslipidemia and inflammation (cholesterol, triglycerides, leptin, MCP-1, IL-1β and IL-6) were increased and KD-fed mice showed signs of hepatic steatosis after 22 weeks of diet. Long-term KD resulted in glucose intolerance that was associated with insufficient insulin secretion from beta cells. After 22 weeks insulin-stimulated glucose uptake was reduced. A reduction in beta cell mass was observed in KD-fed mice together with an increased number of smaller islets. Also alpha cell mass was markedly decreased, resulting in a lower alpha to beta cell ratio. Our data show that long-term KD causes dyslipidemia, a pro-inflammatory state, signs of hepatic steatosis, glucose intolerance and a reduction in beta and alpha cell mass, but no weight loss. This indicates that long-term high-fat, low-carbohydrate ketogenic diets lead to features that are also associated with the metabolic syndrome and an increased risk for type 2 diabetes in humans.

    January 07, 2014   doi: 10.1152/ajpendo.00453.2013   open full text
  • Leanness and Heightened Non-Resting Energy Expenditure: Role of Skeletal Muscle Activity Thermogenesis.
    Gavini, C. K., Mukherjee, S., Shukla, C., Britton, S. L., Koch, L. G., Shi, H., Novak, C. M.
    AJP Endocrinology and Metabolism. January 07, 2014

    A high-calorie diet accompanied by low levels of physical activity (PA) accounts for the widespread prevalence of obesity today. Yet, some people remain lean even in this obesogenic environment. Here, we investigate the cause for this exception. A key trait that predicts high PA in both humans and laboratory rodents is intrinsic aerobic capacity. Rats artificially selected as high capacity runners (HCR) are lean and consistently more physically active than their low- capacity runner (LCR) counterparts; this applies to both males and females. Here, we demonstrate that HCR show heightened total energy expenditure (TEE) and hypothesize that this is due to higher non-resting energy expenditure (NREE). After matching for body weight and lean mass, female HCR consistently had heightened non-resting EE, but not resting EE, compared to female LCR. Because of the dominant role of skeletal muscle in non-resting EE, we examined muscle energy use. We found that lean female HCR had higher muscle heat dissipation during activity, explaining their low economy of activity and high activity EE. This may be due to the amplified skeletal muscle expression levels of proteins involved in EE, and reduced expression levels of proteins involved in energy conservation, in HCR relative to LCR. This is also associated with an increased sympathetic drive to skeletal muscle in HCR compared to LCR. We find little support for the hypothesis that resting metabolic rate is correlated with maximal aerobic capacity if body size and composition are fully considered; rather, the critical factor appears to be activity thermogenesis.

    January 07, 2014   doi: 10.1152/ajpendo.00555.2013   open full text
  • Impaired Exercise-Induced Mitochondrial Biogenesis in the Obese Zucker Rat, Despite PGC-1{alpha} Induction, is Due to Compromised Mitochondrial Translation Elongation.
    Greene, N. P., Nilsson, M. I., Washington, T. A., Lee, D. E., Brown, L. A., Papineau, A. M., Shimkus, K. L., Greene, E. S., Crouse, S. F., Fluckey, J. D.
    AJP Endocrinology and Metabolism. January 07, 2014

    We previously demonstrated that high volume resistance exercise stimulates mitochondrial protein synthesis (a measure of mitochondrial biogenesis) in lean but not obese Zucker rats. Here we examined factors involved in regulating mitochondrial biogenesis in the same animals. PGC-1α was 45% higher following exercise in obese, but not lean animals, when compared to sedentary counterparts. Interestingly, exercised animals demonstrated greater PPAR protein in both lean (47%) and obese (>200%) animals. AMPK phosphorylation (300%) and CPT-I protein (30%) were elevated by exercise in lean animals only, indicating improved substrate availability/flux. These findings suggest that, despite PGC-1α induction, obese animals were resistant to exercise-induced synthesis of new mitochondrial and oxidative protein. We previously reported that most anabolic processes are up-regulated in these same obese animals regardless of exercise, so the purpose of this study was to assess specific factors associated with the mitochondrial genome as possible culprits for impaired mitochondrial biogenesis. Exercise resulted in higher mRNA contents of mitochondrial transcription factor A (TFAM, ~50% in each phenotype) and mitochondrial translation initiation factor 2 (mtIF2, 31 and 47% in lean and obese, respectively). However, mitochondrial translation elongation factor-Tu (TUFM) mRNA was higher following exercise in lean animals only (40%), suggesting aberrant regulation of mitochondrial translation elongation as a possible culprit in impaired mitochondrial biogenesis following exercise with obesity.

    January 07, 2014   doi: 10.1152/ajpendo.00671.2013   open full text
  • Transgenic Overexpression of Hexose-6-Phosphate Dehydrogenase in Adipose Tissue Causes local Glucocorticoid Amplification and Lipolysis in Male Mice.
    Wang, Y., Liu, L., Du, H., Nagaoka, Y., Fan, W., Lutfy, K., Friedman, T. C., Jiang, M., Liu, Y.
    AJP Endocrinology and Metabolism. December 31, 2013

    The pre-receptor activation of glucocorticoid production in adipose tissue by NADPH-dependent 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) has emerged as a potential mechanism in the pathogenesis of visceral obesity and metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) is an endoplasmic reticulum lumen-resident enzyme that generates cofactor NADPH and thus mediates 11ß-HSD1 activity. To determine the role of adipose H6PDH in the prereceptor modulation of 11ß-HSD1 and metabolic phenotypes, we generated a transgenic (Tg) mouse model overexpressing H6PDH under the control of the enhancer-promoter region of the adipocyte fatty acid binding protein (aP2) gene (aP2/H6PDH Tg mice). Transgenic aP2/H6PDH mice exhibited relatively high expression of H6PDH and elevated corticosterone production with induction of 11ß-HSD1 activity in adipose tissue. This increase in corticosterone production in aP2-H6PDH Tg mice resulted in mild abdominal fat accumulation with induction of C/EBP mRNA expression and slight weight gain. Transgenic aP2/H6PDH mice also exhibited fasting hyperglycemia and glucose intolerance with insulin resistance. In addition, the aP2/H6PDH Tg mice have elevated circulating free fatty acid levels with a concomitant increased adipose lipolytic action associated with elevated HSL mRNA and Ser660 HSL phosphorylation within abdominal fat. These results suggest that increased H6PDH expression specifically in adipose tissue is sufficient to cause intra-adipose GC production and adverse metabolic phenotypes. These findings suggest that the aP2/H6PDH Tg mice may provide a favorable model for studying the potential impact of H6PDH in the pathogenesis of human metabolic syndrome.

    December 31, 2013   doi: 10.1152/ajpendo.00491.2013   open full text
  • Exercise training boosts eNOS-dependent mitochondrial biogenesis in mouse heart: role in adaptation of glucose metabolism.
    Vettor, R., Valerio, A., Ragni, M., Trevellin, E., Granzotto, M., Olivieri, M., Tedesco, L., Ruocco, C., Fossati, A., Fabris, R., Serra, R., Carruba, M. O., Nisoli, E.
    AJP Endocrinology and Metabolism. December 31, 2013

    Endurance exercise training increases cardiac energy metabolism through poorly understood mechanisms. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) in cardiomyocytes contributes to cardiac adaptation. Here we demonstrate that the NO donor diethylenetriamine-NO (DETA-NO) activated mitochondrial biogenesis and function, as assessed by up-regulated peroxisome proliferator-activated receptor coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial transcription factor A (Tfam) expression, and by increased mitochondrial DNA content and citrate synthase activity in primary mouse cardiomyocytes. DETA-NO also induced mitochondrial biogenesis and function, and enhanced both basal and insulin-stimulated glucose uptake in HL-1 cardiomyocytes. The DETA-NO-mediated effects were suppressed by either PGC-1α or Tfam small interference RNA in HL-1 cardiomyocytes. Wild-type and eNOS-/- mice were subjected to 6-wk graduated swim training. We found that eNOS expression, mitochondrial biogenesis, mitochondrial volume density and number, and both basal and insulin-stimulated glucose uptake were increased in left ventricles of swim-trained wild-type mice. On the contrary, the genetic deletion of eNOS prevented all these adaptive phenomena. Our findings demonstrate that exercise training promotes eNOS-dependent mitochondrial biogenesis in heart, that behaves as an essential step in cardiac glucose transport.

    December 31, 2013   doi: 10.1152/ajpendo.00617.2013   open full text
  • Skeletal muscle as a target of LXR agonist after long-term treatment: focus on lipid homeostasis.
    Archer, A., Laurencikiene, J., Ahmed, O., Steffensen, K. R., Parini, P., Gustafsson, J.-A., Korach-Andre, M.
    AJP Endocrinology and Metabolism. December 24, 2013

    The liver X receptors (LXR)α and LXRβ are transcription factors belonging to the nuclear receptor family which play a central role in metabolic homeostasis being master regulators of key target genes in the glucose and lipid pathways. Wild-type (Wt), LXRα-/- and LXRβ-/- mice were fed a chow diet with (treated) or without (control) the synthetic dual LXR agonist GW3965 for 5 weeks. GW3965 raised intra-hepatic triglyceride (Tg) level but, surprisingly, reduced serum Tg level through the activation of serum lipase activity. The serum Tg reduction was associated with a repression of both catecholamine-stimulated lipolysis and relative glucose incorporation into lipid in isolated adipocytes through activation of LXRβ. We also demonstrated that LXRα is required for basal (non-stimulated) adipocyte metabolism while LXRβ acts as a repressor of lipolysis. On the contrary, in skeletal muscle (SM) the lipogenic and the cholesterol transporters LXR target genes were markedly induced in Wt and LXRα-/- mice and to a lower extent in LXRβ-/- mice, following treatment with GW3965. Moreover, Tg content was reduced in SM of LXRβ-/- mice associated with an increased expression of the main Tg-lipase genes Hsl and Atgl. Energy expenditure was increased and a switch from glucose to lipid oxidation was observed. In conclusion, we provide evidence that LXR might be an essential regulator of the lipid balance between tissues to ensure appropriate control of the flux of fuel. Importantly, we show that after chronic treatment with GW3965, SM becomes the target tissue for LXR activation, as opposed to liver in acute treatment.

    December 24, 2013   doi: 10.1152/ajpendo.00410.2013   open full text
  • Nongenomic effects of estrogen mediate the dose-related myocardial oxidative stress and dysfunction caused by acute ethanol in female rats.
    El-Mas, M. M., Abdel-Rahman, A. A.
    AJP Endocrinology and Metabolism. December 24, 2013

    Acute ethanol lowers blood pressure (BP) and cardiac output in proestrus and after chronic estrogen (E2) replacement in ovariectomized (OVX) female rats. However, whether rapid nongenomic effects of estrogen mediate these hemodynamic effects of ethanol remained unanswered. To test this hypothesis, we investigated the effect of ethanol (0.5 or 1.5 g/kg; i.v.) on left ventricular (LV) function and oxidative markers in OVX rats pretreated 30 min earlier with 1 μg/kg E2 (OVXE2) or vehicle (OVX) and in proestrus sham-operated (SO) rats. In SO rats, ethanol caused significant and dose-related reductions in BP, rate of rise in LV pressure (LV dP/dtmax), and LV developed pressure (LVDP). These effects of ethanol disappeared in OVX rats and were restored in OVXE2 rats suggesting rapid estrogen receptor signaling mediates the detrimental effects of ethanol on LV function. Ex vivo studies revealed that the estrogen-dependent myocardial dysfunction caused by ethanol was coupled with higher left ventricular: (i) generation of reactive oxygen species (ROS), (ii) expression of malondialdehyde (MDA) and. 4-hydroxynonenal (4-HNE) protein adducts, (iii) phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2), and (iv) catalase activity. ERK1/2 inhibition by PD98059 (1 mg/kg i.v.) abrogated the myocardial dysfunction, hypotension and the elevation in myocardial ROS generation caused by ethanol. It is concluded that rapid estrogen receptor signaling is implicated in cellular events that lead to the generation of aldehyde protein adducts and Akt/ERK1/2 phosphorylation, which ultimately mediate the estrogen-dependent LV oxidative stress and dysfunction caused by ethanol in female rats.

    December 24, 2013   doi: 10.1152/ajpendo.00465.2013   open full text
  • P21-activated protein kinases and their emerging roles in glucose homeostasis.
    Chiang, Y.-t. A., Jin, T.
    AJP Endocrinology and Metabolism. December 24, 2013

    P21-activated protein kinases (PAKs) are centrally involved in a plethora of cellular processes and functions. Their function as effectors of small GTPases Rac1 and Cdc42 has been extensively studied during the past two decades, particularly in the realms of cell proliferation, apoptosis, and hence tumorigenesis; as well as cytoskeletal remodeling and related cellular events in health and disease. In recent years, a large number of studies have shed light onto the fundamental role of group I PAKs, most notably PAK1, in metabolic homeostasis. In skeletal muscle, PAK1 was shown to mediate the function of insulin on stimulating GLUT4 translocation and glucose uptake, while in pancreatic β cells PAK1 participates in insulin granule localization and vesicle release. Furthermore, we demonstrated that PAK1 mediates the crosstalk between insulin and Wnt/β-catenin signaling pathways, and hence regulates gut proglucagon gene expression and the production of the incretin hormone glucagon-like peptide-1 (GLP-1). The utilization of chemical inhibitors of PAK and the characterization of Pak1-/- mice enabled us to gain mechanistic insights as well as to assess the overall contribution of PAKs in metabolic homeostasis. This review summarizes our current understanding of PAKs, with an emphasis on the emerging roles of PAK1 in glucose homeostasis.

    December 24, 2013   doi: 10.1152/ajpendo.00506.2013   open full text
  • Porcine sst1 can physically interact with other somatostatin receptors and its expression is regulated by metabolic/inflammatory sensors.
    Gahete, M. D., Duran-Prado, M., Delgado-Niebla, E., Garrido, J. J., Rhodes, S. J., Garcia-Navarro, S., Gracia-Navarro, F., Malagon, M. M., Luque, R. M., Castano, J. P.
    AJP Endocrinology and Metabolism. December 24, 2013

    The majority of the biological actions attributed to somatostatin (SST) are thought to be mediated by SST receptor 2 (sst2), the most ubiquitous sst, and, to a lesser extent, by sst5. However, a growing body of evidence suggests a relevant role of sst1 in mediating SST actions in (patho)physiologic situations (i.e. endometriosis, type 2 diabetes). Moreover, sst1 together with sst2 and sst5 are involved in the well-known actions of SST on pituitary somatotropes in pig and primates. Here, we cloned the porcine-sst1 (psst1) and performed a structural and functional characterization using both primary and heterologous models. The psst1 sequence presents the majority of signature motifs shared among G-protein coupled receptors and, specifically, among ssts and, exhibits a high homology with other mammalian sst1, with only minor differences in the amino-terminal domain, reinforcing the idea of an early evolutive divergence between mammalian and non-mammalian sst1s. psst1 is functional in terms of decreasing cAMP levels in response to SST when transfected in heterologous models. The psst1 receptor is expressed in several tissues, and analyses of gene cis elements predict regulation by multiple transcription factors and metabolic stimuli. Finally, psst1 is coexpressed with other sst-subtypes in various tissues and in vitro data demonstrate psst1 can interact with itself forming homodimers and with other ssts forming heterodimers. These data highlight the functional importance of sst1 on the SST-mediated effects and its functional interaction with different ssts, which point out the necessity of exploring the consequences of such interactions.

    December 24, 2013   doi: 10.1152/ajpendo.00587.2013   open full text
  • Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice.
    Zabielski, P., Blachnio-Zabielska, A. U., Lanza, I. R., Gopala, S., Manjunatha, S., Jakaitis, D. R., Persson, X.-M., Gransee, J., Klaus, K. A., Coenen-Schimke, J. M., Jensen, M. D., Nair, K. S.
    AJP Endocrinology and Metabolism. December 24, 2013

    Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids, such as ceramides (Cer) and long chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity and at mitochondrial level stimulates reactive oxygen species production. Here we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%A1C) and muscular LCFa-CoA content. The alternations were accompanied by increase in proteins expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1) and inhibition of insulin signaling cascade by AKTα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression and phosphorylation status of AKT and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoA's and Cer's containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, and proteins implicated in Cer synthesis and muscle insulin sensitivity.

    December 24, 2013   doi: 10.1152/ajpendo.00610.2012   open full text
  • Skeletal muscle mitochondrial uncoupling drives endocrine cross-talk through induction of FGF21 as a myokine.
    Keipert, S., Ost, M., Johann, K., Imber, F., Jastroch, M., Van Schothorst, E. M., Keijer, J., Klaus, S.
    AJP Endocrinology and Metabolism. December 17, 2013

    UCP1-TG mice with ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) are a model of improved substrate metabolism and increased longevity. Analysis of myokine expression showed an induction of fibroblast growth factor 21 (FGF21) in SM, resulting in >5 fold elevated circulating FGF21 in UCP1-TG mice. Despite a reduced muscle mass, UCP1-TG mice showed no evidence for a myopathy or muscle autophagy deficiency but an activation of integrated stress response (ISR: eIF2α/ATF4) in SM. Targeting mitochondrial function in vitro by treating C2C12 myoblasts with the uncoupler FCCP resulted in a dose dependent activation of ISR, associated with increased expression of FGF21 which was also observed by treatment with respiratory chain inhibitors antimycin A and myxothiazol. The co-factor required for FGF21 action, β-klotho, was expressed in white adipose tissue (WAT) of UCP1-TG mice which showed an increased browning of WAT, resembled in altered adipocyte morphology, increased brown adipocyte markers (UCP1, CIDEA), lipolysis (HSL phosphorylation), and respiratory capacity. Importantly, treatment of primary white adipocytes with serum of UCP1-TG mice resulted in increased UCP1 expression. Additionally, UCP1-TG mice showed reduced body length through suppressed IGF1/GH axis, and decreased bone mass. We conclude that the induction of FGF21 as a myokine is coupled to disturbance of mitochondrial function and ISR activation in SM. Endocrine acting FGF21 released from SM has endocrine effects leading to increased browning of WAT and can explain the healthy metabolic phenotype of UCP1-TG mice. These results confirm muscle as an important endocrine regulator of whole body metabolism.

    December 17, 2013   doi: 10.1152/ajpendo.00330.2013   open full text
  • G-protein coupled receptor 120 (GPR120) signaling regulates ghrelin secretion in vivo and in vitro.
    Gong, Z., Yoshimura, M., Aizawa, S., Kurotani, R., Zigman, J. M., Sakai, T., Sakata, I.
    AJP Endocrinology and Metabolism. November 12, 2013

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is predominantly produced in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding, and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines, and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by approximately 50 % and 70 %, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW9508. In contrast, the expression levels of prohormone convertase 1 (PC1) were significantly decreased by GW9508 incubation. Moreover, we observed that the inhibitory effect of GW9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Further, pretreatment with GW9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW9508 inhibited ghrelin secretion via extracellular signal-regulated kinase (ERK) activity in ghrelin cell lines. Finally, we found that GW9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is partially induced by long chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.

    November 12, 2013   doi: 10.1152/ajpendo.00306.2013   open full text
  • PAT1 (SLC36A1) shows nuclear localization and affect growth of smooth muscle cells from rats.
    Jensen, A., Figueiredo-Larsen, M., Holm, R., Larsen Broberg, M., Brodin, B., Nielsen, C. U.
    AJP Endocrinology and Metabolism. November 12, 2013

    The proton-coupled amino acid transporter 1 (PAT1) is a transporter of amino acids in small intestinal enterocytes. PAT1 is, however, also capable of regulating cell growth and sensing the availability of amino acids in other cell types. The aim of the present study was to investigate the localization and function of PAT1 in smooth muscle cells (SMCs). The PAT1 protein was found in smooth muscles from rat intestine and in the embryonic rat aorta cell line A7r5. Immunolocalization and cellular fractionation studies revealed that the majority of the PAT1 protein located within the cell nucleus of A7r5. These results were confirmed in primary SMCs derived from rat aorta and colon. A 3'-untranslated region of the PAT1 transcript directed the nuclear localization. Neither cellular starvation nor cell division altered the nuclear localization. In agreement, uptake studies of L-proline, a PAT1 substrate, in A7r5 cells suggested an alternative role for PAT1 in SMCs than in transport. To shed light on the function of PAT1 in A7r5 cells, experiments with down regulation of the PAT1 level using a siRNA approach were conducted. The growth rates of the cells were evaluated and knock-down of PAT1 lead to induced cellular growth suggesting a role for PAT1 in regulating cellular proliferation of SMCs.

    November 12, 2013   doi: 10.1152/ajpendo.00322.2013   open full text
  • Paradoxical resistance to high fat diet-induced obesity and altered macrophage polarization in mineralocorticoid receptor overexpressing mice.
    Kuhn, E., Bourgeois, C., Keo, V., Viengchareun, S., Muscat, A., Meduri, G., Le Menuet, D., Feve, B., Lombes, M.
    AJP Endocrinology and Metabolism. November 12, 2013

    The mineralocorticoid receptor (MR) exerts pro-adipogenic and anti-thermogenic effects in vitro, yet its in vivo metabolic impact remains elusive. Wild type (WT) and transgenic (Tg) mice overexpressing human MR were subjected to standard chow (SC) or high fat diet (HFD) for 16 wks. Tg mice had a lower body weight gain than WT animals, and exhibited a relative resistance to HFD-induced obesity. This was associated with a decrease of fat mass, an increased population of smaller adipocytes, and an improved glucose tolerance compared to WT animals. Quantitative RT-PCR studies revealed decreased expression of PPAR2, a master adipogenic gene, and of glucocorticoid receptor and 11β-hydroxysteroid dehydrogenase type 1, consistent with an impaired local glucocorticoid signaling in adipose tissues (AT). This paradoxical resistance to HFD-induced obesity was not related to an adipogenesis defect since differentiation capacity of Tg preadipocytes isolated from stroma-vascular fractions was unaltered, suggesting that other non-adipocyte factors might compromise AT development. While AT macrophage infiltration was not different between genotypes, Tg mice exhibited a distinct macrophage polarization as revealed by FACS analysis and CD11c/CD206 expression studies. We further demonstrated that Tg macrophage-conditioned medium partially impaired preadipocyte differentiation. Therefore we propose that modification of M1/M2 polarization of hMR-overexpressing macrophages could account in part for the metabolic phenotype of Tg mice. Collectively, our results provide evidence that MR exerts a pivotal immunometabolic role by directly controlling adipocyte differentiation processes but also indirectly through macrophage polarization regulation. Our findings should be taken into account for the pharmacological treatment of metabolic disorders.

    November 12, 2013   doi: 10.1152/ajpendo.00323.2013   open full text
  • Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo.
    Chen, G., Yao, C., Hasegawa, T., Akamatsu, T., Yoshimura, H., Hosoi, K.
    AJP Endocrinology and Metabolism. November 05, 2013

    In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (i.p.) of isoproterenol (IPR), a β-adrenergic agonist, at 1 h, and stayed at high level until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR, the AQP5 mRNA gradually increased and reached a maximal at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR followed by its degradation/metabolism by activation of proteolytic systems. Pre-treatment of animals with 2 calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by 2 proteasome inhibitors, MG132 and lactacystin, and a lysosome denaturant, chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was degraded also by μ-calpain in vitro. Furthermore, we demonstrated that μ-calpain was co-localized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

    November 05, 2013   doi: 10.1152/ajpendo.00317.2013   open full text
  • Protein Synthesis in Skeletal Muscle of Neonates Is Enhanced by Administration of {beta}-Hydroxy-{beta}-Methylbutyrate.
    Wheatley, S. M., El-Kadi, S. W., Suryawan, A., Boutry, C., Orellana, R. A., Nguyen, H. V., Davis, S. R., Davis, T. A.
    AJP Endocrinology and Metabolism. November 05, 2013

    Many low birth weight infants experience failure to thrive. The amino acid, leucine, stimulates protein synthesis in skeletal muscle of the neonate but less is known about the effects of the leucine metabolite, β-hydroxy-β-methylbutyrate (HMB). To determine the effects of HMB on protein synthesis and the regulation of translation initiation and degradation pathways, overnight fasted neonatal pigs were infused with HMB at 0, 20, 100, or 400 µmol•kg body weight (BW)-1•h-1 for 1 h (HMB 0, HMB 20, HMB 100, or HMB 400). Plasma HMB concentrations increased with infusion and were 10, 98, 316, and 1400 nmol•ml-1 in the HMB 0, HMB 20, HMB 100, and HMB 400 pigs. Protein synthesis rates in the longissimus dorsi (LD), gastrocnemius, soleus, and diaphragm muscles, lung, and spleen were greater in HMB 20 than HMB 0 and in the LD were greater in HMB 100 than HMB 0. HMB 400 had no effect on protein synthesis. Eukaryotic initiation factor (eIF) 4E•eIF4G complex formation and ribosomal protein S6 kinase-1 and 4E-binding protein 1 phosphorylation increased in LD, gastrocnemius, and soleus muscles with HMB 20 and HMB 100 and in diaphragm with HMB 20. Phosphorylation of eIF2α and elongation factor 2 and expression of system A transporter (SNAT2), system L transporter (LAT1), muscle RING finger 1 protein (MuRF1), muscle atrophy F-box (atrogin-1), and microtubule-associated protein light chain 3 (LC3-II) were unchanged. Results suggest supplemental HMB enhances protein synthesis in skeletal muscle of neonates by stimulating translation initiation.

    November 05, 2013   doi: 10.1152/ajpendo.00500.2013   open full text
  • Effect of peripherally administered leptin antagonist on whole-body metabolism, and bone microarchitecture and biomechanical properties in mouse.
    Solomon, G., Atkins, A., Shahar, R., Gertler, A., Monsonego-Ornan, E.
    AJP Endocrinology and Metabolism. October 29, 2013

    Leptin's in-vivo effect on the rodent skeleton depends on the model used and the mode of administration. Superactive mouse leptin antagonist (SMLA) was produced and then pegylated (PEG) to prolong and enhance its in-vivo activity. We blocked leptin signaling by injecting this antagonist peripherally into normal mice at various time points, and studied their metabolic and skeletal phenotypes. Subcutaneous PEG-SMLA injections into 4-week-old female C57BL/6J mice significantly increased weight gain and food consumption after only 1 month, and the effect lasted for the 3 months of the experiment, proving its central inhibiting activity. Mice showed a significant increase in serum glucose, cholesterol, triglycerides, insulin and homeostasis assessment model-insulin resistance throughout the experiment. Quantification of gene expression in "metabolic" tissues also indicated the development of insulin resistance. Bone analyses revealed a significant increase in trabecular and cortical parameters measured in both the lumbar vertebrae and tibiae, in PEG-SMLA-treated mice in the first and third months, as well as a significant increase in tibia biomechanical parameters. Interestingly, 30 days of treatment with the antagonist in older mice (aged 3 and 6 months) affected body weight and eating behavior as in the 1-month-old mice, but had no effect on bone parameters, suggesting that leptin's effecton bones, either directly or through its obesogenic effect, is dependent upon stage of skeletal development. This potent and reversible antagonist enabled study leptin's in-vivo role in whole-body and bone metabolism and holds potential for future therapeutic use in diseases involving leptin signaling.

    October 29, 2013   doi: 10.1152/ajpendo.00155.2013   open full text
  • Agouti-Related Peptide Plays a Critical Role in Leptin's Effects on Female Puberty and Reproduction.
    Sheffer-Babila, S., Sun, Y., Israel, D. D., Liu, S.-M., Neal-Perry, G. S., Chua, S. C.
    AJP Endocrinology and Metabolism. October 29, 2013

    Deficient leptin signaling causes infertility via reduced activity of GnRH neurons causing a hypogonadal state in both rodents and humans. AGRP/NPY neurons within the hypothalamic arcuate nucleus are considered to be important intermediate neurons involved in leptin regulation of GnRH neurons. We previously reported that absence of AGRP and haploinsufficiency of MC4R in leptin receptor mutant (Lepr db/db) females restored fertility and lactation despite persistence of obesity and insulin resistance. The overarching hypothesis in the present study is that the absence or reduction of leptin's inhibition of AGRP/NPY neurons leads to suppression of GnRH release in cases of leptin signaling deficiency. Since TAC2 signaling plays a role in pubertal maturation and modulated by metabolic status, this study additionally hypothesized that TAC2 neurons may be regulated by melanocortinergic signals as part of leptin's action on female puberty and reproduction. Our data show that AGRP deficiency in Lepr db/db females restores normal timing of vaginal opening and estrous cycling although uterine weight gain and mammary gland development are morphologically delayed. Nonetheless, Agrp -/- Lepr db/db females are fertile and sustain adequate nutrition of pups with lactation to weaning age. AGRP deficiency results in advanced vaginal opening in wild-type female mice. Agrp -/- Lepr db/db rather Lepr db/db females presented the post-pubertal increase in hypothalamic Tac2 mRNA expression. Furthermore, MC4R activation with MTII induced FOS expression in TAC2 neurons. These studies suggest that AGRP imposes inhibitory effects on female puberty and reproduction, and that TAC2 neurons may transmit melanocortinergic inhibition of GnRH neurons.

    October 29, 2013   doi: 10.1152/ajpendo.00241.2013   open full text
  • Epinephrine Depletion Exacerbates The Fasting Induced Protein Breakdown In Fast Twitch Skeletal Muscles.
    Graca, F. A., Goncalves, D. A., Silveira, W. A., Lira, E. C., Chaves, V. E., Zanon, N. M., Garofalo, M. A. R., Kettelhut, I. C., Navegantes, L. C. C.
    AJP Endocrinology and Metabolism. October 29, 2013

    The physiological role of epinephrine in the regulation of skeletal muscle protein metabolism under fasting is unknown. We examined the effects of plasma epinephrine depletion, induced by adrenodemedullation (ADMX), on muscle protein metabolism in fed and 2-day fasted rats. In fed rats, ADMX for 10 days reduced muscle mass, the cross-sectional area of extensor digitorum longus (EDL) muscle fibers, and the phosphorylation levels of Akt. In addition, ADMX led to a compensatory increase in muscle sympathetic activity, as estimated by the rate of norepinephrine turnover; this increase was accompanied by high rates of muscle protein synthesis. In fasted rats, ADMX exacerbated fasting-induced proteolysis in EDL but did not affect the low rates of protein synthesis. Accordingly, ADMX activated lysosomal proteolysis and further increased the activity of the Ub-proteasome system (UPS). Moreover, expression of the atrophy-related Ub ligases atrogin-1 and MuRF1 and the autophagy-related genes LC3b and GABARAPl1 were up-regulated in EDL muscles from ADMX-fasted rats as compared to sham-fasted rats, and ADMX reduced cAMP levels and increased fasting-induced Akt dephosphorylation. Unlike that observed for EDL muscles, soleus muscle proteolysis and Akt phosphorylation levels were not affected by ADMX. In isolated EDL, epinephrine reduced the basal UPS activity and suppressed overall proteolysis and atrogin-1 and MuRF1 induction following fasting. These data suggest that epinephrine released from the adrenal medulla inhibits fasting-induced protein breakdown in fast-twitch skeletal muscles, and these anti-proteolytic effects on the UPS and lysosomal system are apparently mediated through a cAMP/Akt-dependent pathway, which suppress ubiquitination and autophagy.

    October 29, 2013   doi: 10.1152/ajpendo.00267.2013   open full text
  • Divergent Compensatory Responses to High Fat Diet Between C57BL6/J and C57BLKs/J Inbred Mouse Strains.
    Sims, E. K., Hatanaka, M., Morris, D. L., Tersey, S. A., Kono, T., Chaudry, Z. Z., Day, K. H., Moss, D. R., Stull, N. D., Mirmira, R. G., Evans-Molina, C.
    AJP Endocrinology and Metabolism. October 29, 2013

    Impaired glucose tolerance (IGT) and type 2 diabetes (T2DM) are polygenic disorders with complex pathophysiologies; recapitulating them with mouse models is challenging. Despite 70% genetic homology, C57BL/6J (BL6) and C57BL/KsJ (BLKS) inbred mouse strains differ in response to diet- and genetic-induced obesity. We hypothesized these differences would yield insight into IGT and T2DM susceptibility and response to pharmacological therapies. To this end, male 8 week-old BL6 and BLKS mice were fed normal chow (18% kcal from fat), high fat diet (HFD) (42% kcal from fat), or HFD supplemented with the PPAR- agonist pioglitazone (PIO) (140 mg PIO/kg diet) for 16 weeks. Assessments of body composition, glucose homeostasis, insulin production, and energy metabolism, as well as histologic analyses of pancreata were undertaken. BL6 mice gained weight and adiposity in response to HFD, leading to peripheral insulin resistance that was met with increased β cell proliferation and insulin production. By contrast, BLKS mice responded to HFD by restricting food intake and increasing activity. These behavioral responses limited weight gain and protected against HFD-induced glucose intolerance, which in this strain was primarily due to β cell dysfunction. PIO treatment did not affect HFD-induced weight gain in BL6 mice, and decreased visceral fat mass, while in BLKS mice, PIO increased total fat mass without improving visceral fat mass. Differences in these responses to HFD and effects of PIO reflect divergent human responses to a Western lifestyle and underscore the careful consideration needed when choosing mouse models of diet-induced obesity and diabetes treatment.

    October 29, 2013   doi: 10.1152/ajpendo.00366.2013   open full text
  • Intrahypothalamic pituitary adenylate cyclase-activating polypeptide regulates energy balance via site-specific actions on feeding and metabolism.
    Resch, J. M., Maunze, B., Gerhardt, A. K., Magnuson, S. K., Phillips, K. A., Choi, S.
    AJP Endocrinology and Metabolism. October 22, 2013

    Numerous studies have demonstrated that both the hypothalamic paraventricular nuclei (PVN) and ventromedial nuclei (VMN) regulate energy homeostasis through behavioral and metabolic mechanisms. Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are abundantly expressed in these nuclei suggesting PACAP may be critical for the regulation of feeding behavior and body weight. To characterize the unique behavioral and physiological responses attributed to select hypothalamic cell groups, PACAP was site-specifically injected into the PVN or VMN. Overall food intake was significantly reduced by PACAP at both sites, however meal pattern analysis revealed only injections into the PVN produced significant reductions in meal size, duration, and total time spent eating. PACAP-mediated hypophagia in both the PVN and VMN was abolished by PAC1R antagonism, whereas pretreatment with a VPACR antagonist had no effect. PACAP injections into the VMN produced unique changes in metabolic parameters, including significant increases in core body temperature and spontaneous locomotor activity that was PAC1R dependent whereas, PVN injections of PACAP had no effect. Finally, PACAP-containing afferents were identified using the neuronal tracer, cholera toxin subunit B (CTB), injected unilaterally into the PVN or VMN. CTB signal from PVN injections was co-localized with PACAP mRNA in the medial anterior bed nucleus of the stria terminalis (BNST), VMN, and lateral parabrachial nucleus (LPB). Whereas CTB signal from VMN injections was highly co-localized with PACAP mRNA in the medial amygdala (MeA) and LPB. These brain regions are known to influence energy homeostasis perhaps, in part, through PACAP projections to the PVN and VMN.

    October 22, 2013   doi: 10.1152/ajpendo.00293.2013   open full text
  • Effect of the glucagon-like peptide-1 receptor agonist lixisenatide on postprandial hepatic glucose metabolism in the conscious dog.
    Moore, M. C., Werner, U., Smith, M. S., Farmer, T. D., Cherrington, A. D.
    AJP Endocrinology and Metabolism. October 22, 2013

    The impact of the GLP-1 receptor agonist lixisenatide on postprandial glucose disposition was examined in conscious dogs, to identify mechanisms for its improvement of meal tolerance in humans and examine the tissue disposition of meal-derived carbohydrate. Catheterization for measurement of hepatic balance occurred 16d before study. After fasting overnight, dogs received a subcutaneous injection of lixisenatide 1.5 µg/kg or vehicle (saline; Control), n=6/group. Thirty minutes later, they received an oral meal feeding (93.4 kJ; 19% protein, 71% glucose polymers, and 10% lipid). Acetaminophen was included in the meal in 4 Control and 5 Lixisenatide dogs for assessment of gastric emptying. Observations continued for 510 min; absorption was incomplete in Lixisenatide at that point. The plasma acetaminophen AUC in Lixisenatide was 65% of that in Control (P<0.05). Absorption of the meal began within 15 min in Control but was delayed until 30-45 min in Lixisenatide. Lixisenatide reduced (P<0.05) the postprandial arterial glucose AUC 54% and insulin AUC 44%. Net hepatic glucose uptake did not differ significantly between groups. Nonhepatic glucose uptake tended to be reduced by lixisenatide (6151±4321 and 10541±1854 µmol/kg•510 min in Lixisenatide and Control, P=0.09), but adjusted (for glucose and insulin concentrations), values did not differ (18.9±3.8 and 19.6±7.9 l/kg÷pmol/l, Lixisenatide and Control, respectively; P=0.94). Thus, lixisenatide delays gastric emptying, allowing more efficient disposal of the carbohydrate in the feeding, without increasing liver glucose disposal. Lixisenatide could prove to be a valuable adjunct in treatment of postprandial hyperglycemia in impaired glucose tolerance or type 2 diabetes.

    October 22, 2013   doi: 10.1152/ajpendo.00354.2013   open full text
  • Effects of the Green Tea Polyphenol, Epigallocatechin-3-Gallate (EGCG), on High Fat Diet-Induced Insulin Resistance and Endothelial Dysfunction.
    Jang, H.-J., Ridgeway, S. D., Kim, J.-a.
    AJP Endocrinology and Metabolism. October 22, 2013

    Insulin resistance, a hallmark of metabolic disorders, is a risk factor for diabetes and cardiovascular disease. Impairment of insulin responsiveness in vascular endothelium contributes to insulin resistance. The reciprocal relationship between insulin resistance and endothelial dysfunction augments the pathophysiology of metabolism and cardiovascular functions. The most abundant green tea polyphenol, epigallocatechin-3-gallate (EGCG), has been shown to have vasodilator action in vessels by activation of endothelial nitric oxide synthase (eNOS). However, it is not known whether EGCG has a beneficial effect in high fat diet (HFD)-induced endothelial dysfunction. Male C57BL/6J mice were fed either a normal chow diet (NCD) or HFD with or without EGCG supplement (50 mg/kg/day) for 10 weeks. Mice fed a HFD with EGCG supplement gained less body weight and showed improved insulin sensitivity. In vehicle treated HFD mice, endothelial function was impaired in response to insulin but not to acetylcholine, while EGCG treated HFD group showed improved insulin-stimulated vasodilation. Interestingly, EGCG intake reduced macrophage infiltration into aortic tissues in HFD mice. Pre-treatment with EGCG restored the insulin-stimulated phosphorylation of eNOS, insulin receptor substrate-1 (IRS-1) and protein kinase B (Akt), which was inhibited by palmitate (200 μM, 5 hr) in primary bovine aortic endothelial cells. From these results, we conclude that supplementation of EGCG improves glucose tolerance, insulin sensitivity and endothelial function. The results suggest that EGCG may have beneficial health effects in glucose metabolism and endothelial function through modulating HFD-induced inflammatory response.

    October 22, 2013   doi: 10.1152/ajpendo.00434.2013   open full text
  • Thrombospondin-1/CD36 Pathway Contributes to Bone Marrow-Derived Angiogenic Cell Dysfunction in Type 1 Diabetes via Sonic Hedgehog Pathway Suppression.
    Wang, J.-M., Isenberg, J. S., Billiar, T. R., Chen, A. F.
    AJP Endocrinology and Metabolism. October 22, 2013

    Refractory wounds in diabetic patients present a significant clinical problem. Sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes. Regulation of SHH in wound healing is poorly understood. We hypothesize that thrombospondin-1 (TSP-1), through its receptor CD36, contributes to the SHH signaling defect in bone marrow-derived angiogenic cells (BMACs) in type 1 diabetic mice. Isolated BMACs from TSP-1 knockout mice demonstrated improved tube formation, migration, and adhesion, in parallel with active SHH signaling. BMACs from STZ-induced type 1 diabetic mice showed significantly impaired Matrigel tube formation (n=5, p<0.05 vs. control), which was rescued by TSP-1 depletion (n=5, p<0.05 STZ-TSP-1-/- vs. STZ-WT) or exogenous SHH (20 mg/L, 24 hours, n=4, p<0.05 vs. STZ-control). The expression of CD36 was elevated in BMACs from STZ mice (n=4, p<0.05). SHH signaling was significantly higher in BMACs from TSP-1-/- mice and TSP-1 receptor CD36 knockout mice (n=6, p<0.05 vs. WT), but not CD47 knockout mice (n=3, p>0.05 vs. WT). The impairment of recombinant human TSP-1 (rhTSP-1, 2.2nM, 24h) on BMAC Matrigel tube formation was significantly delayed by CD36 deletion (n=5, p<0.05). CD36-/- BMACs demonstrated better tube formation under both normal and diabetic conditions, with active SHH signaling (n=4, p<0.05 vs. WT BMACs). In conclusion, The TSP-1/CD36 pathway contributes to the SHH signaling defect, resulting in BMAC dysfunction in type 1 diabetic mice.

    October 22, 2013   doi: 10.1152/ajpendo.00516.2013   open full text
  • Sestrin 3 Regulation in Type 2 Diabetic Patients and its Influence on Metabolism and Differentiation in Skeletal Muscle.
    Nascimento, E. B. M., Osler, M. E., Zierath, J. R.
    AJP Endocrinology and Metabolism. October 15, 2013

    In mammals, the sestrin family is composed of 3 stress responsive genes. Ablation of sestrin in Drosophila attenuates longevity, which is accompanied by increased S6K phosphorylation and decreased AMPK phosphorylation. Nevertheless, the metabolic role of sestrins in mammals is not comprehensively understood. We characterized the expression of individual sestrin family members and determined their role in vastus lateralis muscle biopsies from participants with normal glucose tolerance (NGT) or type 2 diabetes (T2D). Expression of sestrin 1 or sestrin 2 mRNA was unaltered between the NGT and T2D participants. Conversely, sestrin 3 mRNA was increased in T2D patients and correlated with fasting plasma glucose, 2 hour post prandial plasma glucose and HbA1c. A trend for increased sestrin 3 protein was observed in T2D patients. In human primary myotubes, sestrin 3 mRNA increased during differentiation and this response was unaltered in T2D-derived myotubes. Long-term treatment of myotubes with insulin or AICAR decreased sestrin 3 mRNA. Exposure of myotubes to the reactive oxygen species hydrogen peroxide increased mRNA expression of sestrin 1 and 2, whereas sestrin 3 was unaltered. siRNA-mediated silencing of sestrin 3 in myotubes was without effect on insulin-stimulated glucose incorporation into glycogen or AICAR-stimulated palmitate oxidation. These results provide evidence against sestrin 3 in the direct control of glucose or lipid metabolism in human skeletal muscle. However, siRNA-mediated sestrin 3 gene silencing in myotubes increased myostatin expression. Collectively, our results indicate sestrin 3 is upregulated in T2D and could influence skeletal muscle differentiation, without altering glucose and lipid metabolism.

    October 15, 2013   doi: 10.1152/ajpendo.00212.2013   open full text
  • Eplerenone ameliorates the phenotypes of metabolic syndrome with NASH in liver-specific SREBP1c Tg mice fed high-fat and high-fructose diet.
    Wada, T., Miyashita, Y., Sasaki, M., Aruga, Y., Nakamura, Y., Ishii, Y., Sasahara, M., Kanasaki, K., Kitada, M., Koya, D., Shimano, H., Tsuneki, H., Sasaoka, T.
    AJP Endocrinology and Metabolism. October 15, 2013

    Because the renin-angiotensin-aldosterone system has been implicated in the development of insulin resistance and promotion of fibrosis in some tissues, such as the vasculature, we examined the effect of eplerenone, a selective mineralocorticoid receptor (MR) antagonist, on non-alcoholic steatohepatitis (NASH) and metabolic phenotypes in a mouse model reflecting metabolic syndrome in humans. We adopted liver-specific transgenic mice overexpressing the active form of sterol response element binding protein 1c (SREBP1c) fed a high-fat and fructose diet (HFFD) as the animal model in the present study. When wild-type (WT) C57BL/6 and liver-specific SREBP1c transgenic (Tg) mice grew while being fed HFFD for 12 weeks, body weight and epididymal fat weight increased in both groups with an elevation in blood pressure and dyslipidemia. Glucose intolerance and insulin resistance were also observed. Adipose tissue hypertrophy and macrophage infiltration with crown-like structure formation were also noted in mice fed HFFD. Interestingly, the changes noted in both genotypes fed HFFD were significantly ameliorated with eplerenone. HFFD-fed Tg mice exhibited the histological features of NASH in the liver, including macrovesicular steatosis and fibrosis, whereas HFFD-fed WT mice had hepatic steatosis without apparent fibrotic changes. Eplerenone effectively ameliorated these histological abnormalities. Moreover, the direct suppressive effects of eplerenone on lipopolysaccharide-induced TNFalpha production in the presence and absence of aldosterone were observed in primary-cultured Kupffer cells and bone marrow-derived macrophages. These results indicated that eplerenone prevented the development of NASH and metabolic abnormalities in mice by inhibiting inflammatory responses in both Kupffer cells and macrophages.

    October 15, 2013   doi: 10.1152/ajpendo.00419.2013   open full text
  • Adiponectin Inhibits Oxidative/Nitrative Stress during Myocardial Ischemia and Reperfusion via PKA Signaling.
    Zhang, Y., Wang, X.-L., Zhao, J., Wang, Y.-J., Lau, W. B., Yuan, Y.-X., Gao, E.-H., Koch, W. J., Ma, X.-L.
    AJP Endocrinology and Metabolism. October 15, 2013

    The cardioprotective effects of adiponectin (APN) against myocardial ischemia/reperfusion (MI/R) injury are well known. However, comprehension of the mechanisms mediating intracellular APN signaling remains incomplete. We recently demonstrate the antioxidant/antinitrative effects of APN are not dependent upon AMPK. Protein Kinase A (PKA) has been previously shown to be activated by APN, with uncertain relevance to APN cardiac protection. The current study determined whether the anti-oxidative/anti-nitrative effect of APN is mediated by PKA. Administration of APN (2 µg/g) 10 minutes before reperfusion significantly enhanced cardiac PKA activity, reduced oxidative stress and decreased infarct size. Knockdown of cardiac PKA expression (PKA-KD) by intramyocardial injection of PKA-siRNAs (>70% suppression) significantly inhibited APN cardioprotection determined by cardiac apoptosis, infarct size, and cardiac function. Moreover, PKA-KD virtually abolished the suppressive effect of APN on MI/R induced NADPH oxidase overexpression and superoxide overproduction, and partially inhibited the effect of APN on nitrative protein modification in MI/R heart. Mechanistically, APN significantly inhibited MI/R-induced IKK/IB phosphorylation and NFB activation, which were blocked in PKA-KD heart. Finally, the PKA-mediated antioxidant/antinitrative and cardioprotective effects of APN are intact in AMPK deficiency mice, suggesting that there is no cross-talk between AMPK and PKA signaling in APN cardioprotection. Collectively, we demonstrate for the first time that APN inhibits oxidative/nitrative stress during MI/R via PKA-dependent NFB inhibition.

    October 15, 2013   doi: 10.1152/ajpendo.00445.2013   open full text
  • Determinants of Reversibility of Beta-cell Dysfunction in response to Short-term Intensive Insulin Therapy in Patients with early Type 2 Diabetes.
    Kramer, C. K., Choi, H., Zinman, B., Retnakaran, R.
    AJP Endocrinology and Metabolism. October 15, 2013

    Short-term intensive insulin therapy (IIT) can improve pancreatic beta-cell function when administered early in the course of type 2 diabetes (T2DM). However, the degree of improvement in response to this therapy varies between patients. Thus, we sought to characterize the determinants of improvement in beta-cell function in response to short-term IIT in early T2DM. Sixty-three patients with mean 3.0±2.1 years duration of T2DM and HbA1c 6.8±0.8% underwent 4 weeks of IIT consisting of basal insulin detemir and pre-meal insulin aspart, with oral glucose tolerance test administered at baseline and 1-day post-IIT. Beta-cell function before and after IIT was assessed by Insulin Secretion-Sensitivity Index-2 (ISSI-2). Reversibility of beta-cell dysfunction was defined as percentage change in ISSI-2 ≥25%. Overall, the study population experienced an increase in ISSI-2 from baseline to post-IIT (P=0.01), with one third of participants achieving ≥25% improvement in ISSI-2. Compared to their peers, those with increase in ISSI-2 ≥25% had greater decrements in fasting glucose (P<0.0001), HbA1c (P=0.001), ALT (P=0.04), AST (P=0.02), and HOMA-IR (P<0.0001). On logistic regression analysis, baseline HbA1c (OR=2.83, 95%CI 1.16-6.88, P=0.02) and change in HOMA-IR (OR=0.008, 95%CI 0.0004-0.16, P=0.001) emerged as independent predictors of reversibility of beta-cell dysfunction. Indeed, reversibility of beta-cell dysfunction was achieved in only those participants in whom IIT yielded an improvement in HOMA-IR. In conclusion, decline in HOMA-IR may be a key determinant of improvement of beta-cell function in response to short-term IIT, suggesting a fundamental contribution of insulin resistance to the reversible component of beta-cell dysfunction in early T2DM.

    October 15, 2013   doi: 10.1152/ajpendo.00447.2013   open full text
  • The Mechanical Properties of Human Adipose Tissues and their Relationships to the Structure and Composition of the Extracellular Matrix.
    Alkhouli, N., Mansfield, J., Green, E., Bell, J., Knight, B., Liversedge, N., Tham, J. C., Welbourn, R., Shore, A. C., Kos, K., Winlove, C. P.
    AJP Endocrinology and Metabolism. October 08, 2013

    Adipose tissue (AT) expansion in obesity is characterized by cellular growth and continuous extracellular matrix (ECM) remodeling, with increased fibrillar collagen deposition. It is hypothesized that the matrix can inhibit cellular expansion and lipid storage. It is therefore important to fully characterize the ECM's biomechanical properties and its interactions with cells. In this study we characterize and compare the mechanical properties of human subcutaneous and omental tissues, which have different physiological functions. AT was obtained from 44 subjects undergoing surgery. Force-extension and stress-relaxation data were obtained. The effects of osmotic challenge were measured to investigate the cellular contribution to tissue mechanics. Tissue structure and its response to tensile strain was determined using nonlinear microscopy. AT showed non-linear stress-strain characteristics up to 30% strain. Comparing paired subcutaneous and omental samples (n=19) the moduli were lower in subcutaneous: initial 1.6±0.8KPa (mean±SD) and 2.9±1.5KPa (p=0.001), final 11.7±6.4KPa and 32±15.6KPa (p<0.001) respectively. The energy dissipation density was lower in subcutaneous AT (n=13): 0.1±0.1KPa and 0.3±0.2KPa respectively (p=0.006). Stress-relaxation followed a two-exponential time course. When the incubation medium was exchanged for deionized water in specimens held at 30% strain, force decreased by 31% and the final modulus significantly increased. Nonlinear microscopy revealed collagen and elastin networks in close proximity to adipocytes and a larger-scale network of larger fiber bundles. There was considerable micro-scale heterogeneity in the response to strain in both cells and matrix fibers. These results suggest that subcutaneous AT has greater capacity for expansion and recovery from mechanical deformation than omental AT.

    October 08, 2013   doi: 10.1152/ajpendo.00111.2013   open full text
  • Autocrine role of interleukin-13 on skeletal muscle glucose metabolism in Type 2 diabetic patients involves microRNA let-7.
    Jiang, L. Q., Franck, N., Egan, B., Sjogren, R. J. O., Katayama, M., Duque-Guimaraes, D., Arner, P., Zierath, J. R., Krook, A.
    AJP Endocrinology and Metabolism. October 08, 2013

    Low-grade inflammation associated with Type 2 Diabetes (T2DM) is postulated to exacerbate insulin resistance. We report that serum levels, as well as IL-13 secreted from cultured skeletal muscle, is reduced in T2DM versus normal glucose tolerant (NGT) subjects. IL-13 exposure increases skeletal muscle glucose uptake, oxidation and glycogen synthesis via an Akt-dependent mechanism. Expression of microRNA let-7a and let-7d, direct translational repressors of the IL-13 gene, was increased in skeletal muscle from T2DM patients. Overexpression of let-7a and let-7d in cultured myotubes reduced IL-13 secretion. Furthermore, basal glycogen synthesis was reduced in cultured myotubes exposed to an IL-13 neutralizing antibody. Thus, IL-13 is synthesized and released by skeletal muscle through a mechanism involving let-7 and this effect is attenuated in skeletal muscle from insulin resistant T2DM patients. In conclusion, IL-13 plays an autocrine role in skeletal muscle to increase glucose uptake and metabolism, suggesting a role in glucose homeostasis in metabolic disease.

    October 08, 2013   doi: 10.1152/ajpendo.00236.2013   open full text
  • Overexpression of NF-{kappa}B p65 in Macrophages Ameliorates Atherosclerosis in ApoE Knockout Mice.
    Ye, X., Jiang, X., Guo, W., Clark, K., Gao, Z.
    AJP Endocrinology and Metabolism. October 08, 2013

    The transcription factor NF-B p65 is a key regulator in the regulation of an inflammatory response and in the pathology of atherosclerosis. However, there is no direct evidence for the role of NF-B in macrophages in the development of atherosclerosis. We investigated whether macrophage overexpression of p65 in ApoE knockout mice could improve atherosclerosis. Transgenic mice overexpressing p65 in macrophages (TG) were generated by crossing fatty acid binding protein 4 (aP2) promoter-controlled p65 mice with ApoE knockout (KO) mice. TG mice exhibited functional activation of NF-B signaling in macrophages and fat tissues. We observed that the atherosclerotic lesion was 40% less in the TG mice compared with the ApoE KO controls fed with a standard atherogenic diet for 16 weeks (N = 12). The TG mice were leaner from reduced fat mass by increased energy expenditure. Moreover, the overexpression of p65 in macrophages suppressed foam cell formation. Our results show that there are 1) an increased fatty acid oxidation in macrophages, 2) a reduced scavenger receptor CD36 expression and lipid accumulation in microphages, 3) reduced inflammation cytokines in serum, and 4) enhanced energy expenditure in TG mice. Our data suggest that activation of NF-B in macrophages has atheroprotective effects in mice by enhancing lipid metabolism and energy expenditure.

    October 08, 2013   doi: 10.1152/ajpendo.00307.2013   open full text
  • Molecular Mechanisms that Drive Estradiol-Dependent Burst Firing of Kiss1 Neurons in the Rostral Periventricular Preoptic Area.
    Zhang, C., Tonsfeldt, K. J., Qiu, J., Bosch, M. A., Kobayashi, K., Steiner, R. A., Kelly, M. J., Ronnekleiv, O. K.
    AJP Endocrinology and Metabolism. October 08, 2013

    Kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3V) provide excitatory drive to gonadotropin-releasing hormone (GnRH) neurons to control fertility. Using whole-cell patch clamp recording and single-cell (sc)RT-PCR techniques targeting Kiss1-CreGFP or TH-EGFP neurons, we characterized the biophysical properties of these neurons and identified the critical intrinsic properties required for burst firing in 17β-estradiol (E2)-treated, ovariectomized female mice. A quarter of the RP3V Kiss1 neurons exhibited spontaneous burst firing. RP3V Kiss1 neurons expressed a hyperpolarization-activated h-current (Ih) and a T-type calcium current (IT), which supported hyperpolarization-induced rebound burst firing. Under voltage clamp conditions, all Kiss1 neurons expressed a kinetically fast Ih that was augmented 3.4 fold by high (LH surge producing) E2 treatment. ScPCR analysis of Kiss1 neurons revealed abundant expression of the HCN1 channel transcripts. Kiss1 neurons also expressed a Ni2+ and TTA-P2 sensitive IT that was augmented 6-fold with high E2 treatment. CaV3.1 mRNA was also highly expressed in these cells. Current clamp analysis revealed that rebound burst firing was induced in RP3V Kiss1 neurons in high E2-treated animals, and the majority of Kiss1 neurons had a hyperpolarization threshold of -84.7 mV, which corresponded to the V1/2 for IT de-inactivation. Finally, Kiss1 neurons in the RP3V were hyperpolarized by µ- and -opioid and GABAB receptor agonists, suggesting that these pathways also contribute to rebound burst firing. Therefore, Kiss1 neurons in the RP3V express the critical channels and receptors that permit E2-dependent rebound burst firing and provide the biophysical substrate that drives the preovulatory surge of GnRH.

    October 08, 2013   doi: 10.1152/ajpendo.00406.2013   open full text
  • The food intake-suppressive effects of glucagon-like peptide-1 receptor signaling in the ventral tegmental area are mediated by AMPA/kainate receptors.
    Mietlicki-Baase, E. G., Ortinski, P. I., Rupprecht, L. E., Olivos, D. R., Alhadeff, A. L., Pierce, R. C., Hayes, M. R.
    AJP Endocrinology and Metabolism. October 08, 2013

    Glucagon-like peptide-1 receptor (GLP-1R) activation in the ventral tegmental area (VTA) is physiologically relevant for the control of palatable food intake. Here, we tested whether the food intake-suppressive effects of VTA GLP-1R activation are mediated by glutamatergic signaling within the VTA. Intra-VTA injections of the GLP-1R agonist exendin-4 (Ex-4) reduced palatable high-fat food intake in rats primarily by reducing meal size; these effects were mediated in part via glutamatergic AMPA/kainate, but not NMDA, receptor signaling. Additional behavioral data indicated that GLP-1R expressed specifically within the VTA can partially mediate the intake- and body-weight-suppressive effects of systemically administered Ex-4, offering the intriguing possibility that this receptor population may be clinically relevant for food intake control. Intra-VTA Ex-4 rapidly increased tyrosine hydroxylase levels within the VTA, suggesting that GLP-1R activation modulates VTA dopaminergic signaling. Further evidence for this hypothesis was provided by electrophysiological data showing that Ex-4 increased the frequency of AMPA-mediated currents and reduced paired-pulse ratio in VTA dopamine neurons. Together, these data provide novel mechanisms by which GLP-1R agonists in the mesolimbic reward system control for palatable food intake.

    October 08, 2013   doi: 10.1152/ajpendo.00413.2013   open full text
  • Mitochondrial fragmentation impairs insulin-dependent glucose uptake by Akt activity through mitochondrial Ca2+ uptake.
    del Campo, A., Parra, V., Vasquez-Trincado, C., Gutierrez, T., Morales, P. E., Lopez-Crisosto, C., Bravo-Sagua, R., Navarro-Marquez, M. F., Verdejo, H. E., Contreras-Ferrat, A., Troncoso, R., Chiong, M., Lavandero, S.
    AJP Endocrinology and Metabolism. October 01, 2013

    Insulin is a major regulator of glucose metabolism, stimulating its mitochondrial oxidation in skeletal muscle. Mitochondria are dynamic organelles that can undergo structural remodeling in order to cope with these ever-changing metabolic demands. However, how mitochondrial morphology impacts insulin signaling in the skeletal muscle is far from being completely elucidated. To address this, we silenced two mitochondrial fusion proteins, Mfn2 and Opa1, and assessed insulin response in L6 rat skeletal muscle cells. We found that mitochondrial fragmentation attenuates insulin-stimulated Akt phosphorylation, glucose uptake, and respiratory rate. Insulin is a major regulator of glucose metabolism, stimulating its mitochondrial oxidation in skeletal muscle cells. Mitochondria are dynamic organelles that can undergo structural remodeling in order to cope with these ever-changing metabolic demands. However, the process by which mitochondrial morphology impacts insulin signaling in the skeletal muscle cells remains uncertain. To address this question, we silenced the mitochondrial fusion proteins Mfn2 and Opa1 and assessed insulin-dependent responses in L6 rat skeletal muscle cells. We found that mitochondrial fragmentation attenuates insulin-stimulated Akt phosphorylation, glucose uptake, and cell respiratory rate. Importantly, we found that insulin induces a transient raise in mitochondrial Ca2+ uptake, which was attenuated by silencing Opa1 or Mfn2. Moreover, treatment with Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, impairs Akt signaling without affecting mitochondrial dynamics. Altogether, these results suggest that control of mitochondrial Ca2+ uptake by mitochondrial morphology is a key event for insulin-induced glucose uptake.

    October 01, 2013   doi: 10.1152/ajpendo.00146.2013   open full text
  • Punicalagin, a polyphenol in pomegranate juice, down regulates p53 and attenuates hypoxia-induced apoptosis in cultured human placental syncytiotrophoblasts.
    Chen, B., Longtine, M. S., Nelson, D. M.
    AJP Endocrinology and Metabolism. October 01, 2013

    Oxidative stress is associated with placental dysfunction and sub-optimal pregnancy outcomes. Therapeutic interventions to limit placental injury from oxidative stress are lacking. Punicalagin is an ellagitannin and a potent antioxidant in pomegranate juice. We showed that both pomegranate juice and punicalagin decrease oxidative stress and apoptosis in cultured syncytiotrophoblasts. p53 is involved in the oxidative stress-induced apoptosis in trophoblasts. We now test the hypothesis that punicalagin limits trophoblast injury in vitro by regulating the levels of p53. We examined the expression of p53, MDM2, p21, HIF1α and selected members of the BCL2 family of proteins in cultured syncytiotrophoblasts exposed to < 1% oxygen in the absence or presence of punicalagin. We found that punicalagin attenuated hypoxia induced apoptosis in syncytiotrophoblasts, as quantified by levels of cleaved PARP. This protective effect was in part mediated by reduced p53 activity shown by decreased expression of p21, lower HIF1α expression, and limited activity of caspases 9 and 3. There was no change in expression of proteins in the BCL2 family, which are also important in apoptosis. The data support a role for down regulation of p53 in the protection of human trophoblasts by punicalagin.

    October 01, 2013   doi: 10.1152/ajpendo.00218.2013   open full text
  • Angiotensin type 2 receptor in pancreatic islets of adult rats: a novel insulinotropic mediator.
    Shao, C. H., Zucker, I. H., Gao, L.
    AJP Endocrinology and Metabolism. October 01, 2013

    In the present study, we evaluated the relative abundance of AT2R protein in various tissues of adult rats. We found that pancreatic islets expressed the highest AT2R protein as compared to all other tissues. Accordingly, we then determined the functional significance of AT2R in the endocrine pancreas in vivo and in vitro experiments by using Angiotensin II alone, Losartan (Los; AT1R antagonist), Compound 21 (C21; AT2R agonist), and PD123319 (PD; AT2R antagonist). Experiments carried out in rats indicated that, (1) Ang II treatment significantly increased plasma insulin concentration (1.51 ± 0.20 vs 0.82 ± 0.14 ng/ml, n = 7, P < 0.05) in the fed state. This insulinotropic effect was further augmented by combined treatment with Ang II plus Los (2.31 ± 0.25 ng/ml, n = 7, p < 0.01). C21 also elevated insulin levels (2.13 ± 0.20 ng/ml, n = 7, P < 0.01), which was completely abolished by PD. (2) Ang II impaired glucose tolerance, whereas Ang II plus Los or C21 improved this function. (3) All treated rats displayed an enhanced insulin secretory response to a glucose challenge. (4) All treated rats displayed up-regulated proinsulin 2 mRNA and insulin protein expression in the pancreas. In in vitro experiments using INS-1E cells and isolated rat islets, we found that AT2R activation significantly improved insulin biosynthesis and secretion. These results suggest that the AT2R functions as an insulinotropic mediator. AT2R and its downstream signaling pathways may be potential therapeutic targets for diabetes.

    October 01, 2013   doi: 10.1152/ajpendo.00286.2013   open full text
  • Ectonucleotidase NTPDase3 is abundant in pancreatic {beta} cells and regulates glucose induced insulin secretion.
    Syed, S. K., Kauffman, A. L., Beavers, L. S., Alston, J. T., Farb, T. B., Ficorilli, J., Macrelo, M. C., Brenner, M. B., Bokvist, K., Barrett, D. G., Efanov, A.
    AJP Endocrinology and Metabolism. October 01, 2013

    Extracellular ATP released from pancreatic β cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. We therefore investigated the expression and role of ectonucleotidases in pancreatic β cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose dependent insulin secretion. Down-regulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic β cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.

    October 01, 2013   doi: 10.1152/ajpendo.00328.2013   open full text
  • Rescue of Heart Lipoprotein Lipase Knockout Mice Confirms a Role for Triglyceride in Optimal Heart Metabolism and Function.
    Khan, R. S., Lin, Y., Hu, Y., Son, N.-H., Bharadwaj, K. G., Palacios, C., Chokshi, A., Ji, R., Yu, S., Homma, S., Schulze, P. C., Tian, R., Goldberg, I. J.
    AJP Endocrinology and Metabolism. October 01, 2013

    Hearts utilize fatty acids as a primary source of energy. The sources of those lipids include free fatty acids and lipoprotein triglycerides. Deletion of the primary triglyceride hydrolyzing enzyme lipoprotein lipase (LpL) leads to cardiac dysfunction. Whether heart LpL knockout (hLpL0) mice are compromised due a deficiency in energetic substrates is unknown. To test whether alternative sources of energy will prevent cardiac dysfunction in hLpL0 mice. Two different models were used to supply non-lipid energy: 1) hLpL0 mice were crossed with mice transgenically expressing GLUT1 in cardiomyocytes to increase glucose uptake into the heart. This cross corrected cardiac dysfunction, reduced cardiac hypertrophy, and increased myocardial ATP. 2) Mice were randomly assigned to a sedentary or training group (swimming) at 3 months of age, which leads to increased skeletal muscle production of lactate. hLpL0 mice had greater expression of the lactate transporter monocarboxylate transporter-1 (MCT-1) and increased cardiac lactate uptake. Compared to hearts from sedentary hLpL0 mice, hearts from trained hLpL0 mice had adaptive hypertrophy and improved cardiac function. We conclude that defective energy intake and not the reduced uptake of fat-soluble vitamins or cholesterol is responsible for cardiac dysfunction in hLpL0 mice. In addition, our studies suggest that adaptations in cardiac metabolism contribute to the beneficial effects of exercise on the myocardium of patients with heart failure.

    October 01, 2013   doi: 10.1152/ajpendo.00349.2013   open full text
  • GLP-1-derived nonapeptide GLP-1(28-36)amide represses hepatic gluconeogenic gene expression and improves pyruvate tolerance in high fat diet fed mice.
    Ip, W., Shao, W., Chiang, Y.-t., Jin, T.
    AJP Endocrinology and Metabolism. October 01, 2013

    Certain "degradation" products of GLP-1 were found to possess beneficial effects on metabolic homeostasis. Here, we investigated the function of the C-terminal fragment of GLP-1, the nonapeptide GLP-1(28-36)amide, in hepatic glucose metabolism. C57BL/6 mice fed with high fat diet (HFD) for 13 wks were i.p. injected with GLP-1(28-36)amide for 6 wks. A significant reduction in body weight gain in response to HFD feeding was observed in GLP-1(28-36)amide-treated mice. GLP-1(28-36)amide administration moderately improved glucose disposal during glucose tolerance test but more drastically attenuated glucose production during pyruvate tolerance test, associated with reduced hepatic expression of gluconeogenic genes Pck1, G6pc, and Ppargc1a. Mice treated with GLP-1(28-36)amide exhibited increased phosphorylation of PKA targets including cAMP response element-binding protein (CREB), ATF-1, and β-catenin. In primary hepatocytes, GLP-1(28-36)amide reduced glucose production and expression of Pck1, G6pc, and Ppargc1a, associated with increased cAMP content and PKA target phosphorylation. These effects were attenuated by PKA inhibition. We suggest that GLP-1(28-36)amide represses hepatic gluconeogenesis involving the activation of components of the cAMP/PKA signaling pathway. This study further confirmed that GLP-1(28-36)amide possesses therapeutic potential for diabetes and other metabolic disorders.

    October 01, 2013   doi: 10.1152/ajpendo.00376.2013   open full text
  • Advancements and challenges in generating accurate animal models of gestational diabetes mellitus.
    Pasek, R. C., Gannon, M.
    AJP Endocrinology and Metabolism. October 01, 2013

    The maintenance of glucose homeostasis during pregnancy is critical to the health and well-being of both the mother and the developing fetus. Strikingly, approximately 7% of human pregnancies are characterized by insufficient insulin production or signaling, resulting in gestational diabetes mellitus (GDM). In addition to the acute health concerns of hyperglycemia, women diagnosed with GDM during pregnancy have an increased incidence of complications during pregnancy, as well as an increased risk of developing type 2 diabetes (T2D) later in life. Furthermore, children born to mothers diagnosed with GDM have increased incidence of perinatal complications including hypoglycemia, respiratory distress syndrome, and macrosomia, as well as an increased risk of being obese or developing T2D as adults. No single environmental or genetic factor is solely responsible for the disease. Instead, a variety of risk factors, including weight, ethnicity, genetics, and family history, contribute to the likelihood of developing GDM, making the generation of animal models that fully recapitulate the disease difficult. Here, we discuss and critique the various animal models that have been generated to better understand the etiology of diabetes during pregnancy and its physiological impacts on both the mother and the fetus. Strategies utilized are diverse in nature, and include the use of surgical manipulation, pharmacological treatment, nutritional manipulation, and genetic approaches in a variety of animal models. The continued development of animal models of GDM is essential for understanding the consequences of this disease as well as providing insights into potential treatments and preventative measures.

    October 01, 2013   doi: 10.1152/ajpendo.00425.2013   open full text
  • Altered Body Composition, and Energy Expenditure but Normal Glucose Tolerance among Humans with a Long-Chain Fatty Acid Oxidation Disorder.
    Gillingham, M. B., Harding, C. O., Schoeller, D. A., Matern, D., Purnell, J. Q.
    AJP Endocrinology and Metabolism. September 24, 2013

    The development of insulin resistance has been associated with impaired mitochondrial fatty acid oxidation (FAO), but the exact relationship between FAO capacity and glucose metabolism continues to be debated. To address this controversy, patients with long-chain 3-hydroxy acylCoA dehydrogenase (LCHAD) deficiency underwent an oral glucose tolerance test (OGTT) and measurement of energy expenditure, body composition, and plasma metabolites. Compared to controls, patients with LCHAD deficiency had a trend toward higher total body fat and extramyocellular lipid deposition, but similar levels of intramyocelluar and intrahepatic lipids. Resting energy expenditure was similar between the groups, but respiratory quotient was higher and total energy expenditure was lower in LCHAD-deficient patients compared to controls. High molecular weight (HMW) adiponectin levels were lower and plasma long-chain acylcarnitines were higher among LCHAD-deficient patients. Fasting and post OGTT levels of glucose, insulin and ghrelin, along with estimates of insulin sensitivity, were the same between the groups. Despite decreased capacity for FAO, lower total energy expenditure and plasma HMW adiponectin, and increased plasma acylcarnitines, LCHAD-deficient patients exhibited normal glucose tolerance. These data suggest that inhibition of the FAO pathway in humans is not sufficient to induce insulin resistance.

    September 24, 2013   doi: 10.1152/ajpendo.00225.2013   open full text
  • Hindbrain noradrenergic input to the hypothalamic PVN mediates the activation of oxytocinergic neurons induced by the satiety factor oleoylethanolamide.
    Romano, A., Potes, C. S., Tempesta, B., Cassano, T., Cuomo, V., Lutz, T., Gaetani, S.
    AJP Endocrinology and Metabolism. September 24, 2013

    Oleoylethanolamide (OEA) is a gut-derived endogenous lipid that stimulates vagal fibres to induce satiety. Our previous work has shown that peripherally administered OEA activates c-fos transcription in the nucleus of the solitary tract (NST) and in the paraventricular nucleus (PVN) where it enhances oxytocin (OXY) expression. The anorexigenic action of OEA is prevented by the i.c.v. administration of a selective OXY receptor antagonist, suggesting a necessary role of OXYergic mediation of OEA's effect. The NST is the source of direct noradrenergic afferent input to hypothalamic OXY neurons, and we therefore hypothesized that the activation of this pathway might mediate OEA effects on PVN neurons. To test this hypothesis, we subjected rats to intra-PVN administration of the toxin saporin (DSAP) conjugated to an antibody against dopamine-beta-hydroxylase (DBH), to destroy hindbrain noradrenergic neurons. In these rats we evaluated the effects of OEA (10 mg/kg, i.p.) on feeding behavior, on c-Fos and OXY immunoreactivity in the PVN, and on OXY immunoreactivity in the posterior pituitary gland. We found that the DSAP lesion completely prevented OEA's effects on food intake, on Fos and OXY expression in the PVN and on OXY immunoreactivity of the posterior pituitary gland; all effects were maintained in sham operated rats. These results support the hypothesis that noradrenergic NST-PVN projections are involved in the activation of the hypothalamic OXY system which mediates OEA's pro-satiety action.

    September 24, 2013   doi: 10.1152/ajpendo.00411.2013   open full text
  • Insulin sensitivity and metabolic flexibility following exercise training among different obese insulin resistant phenotypes.
    Malin, S. K., Haus, J. M., Solomon, T. P. J., Blaszczak, A., Kashyap, S. R., Kirwan, J. P.
    AJP Endocrinology and Metabolism. September 24, 2013

    Impaired fasting glucose (IFG) blunts the reversal of impaired glucose tolerance (IGT) after exercise training. Metabolic inflexibility has been implicated in the etiology of insulin resistance, however, the efficacy of exercise on peripheral and hepatic insulin sensitivity or substrate utilization in adults with IFG, IGT or IFG+IGT is unknown. Twenty-four older (66.7±0.8yr) obese (34.2±0.9kg/m2) adults were categorized as IFG (n=8), IGT (n=8), or IFG+IGT (n=8) according to a 75-gram oral glucose tolerance test (OGTT). Subjects underwent 12-weeks of exercise (60 min/d for 5 d/wk at ~85% HRmax) and were instructed to maintain a eucaloric diet. A euglycemic-hyperinsulinemic clamp (40 mU/m2/min) with [6,6-2H]-glucose was used to determine peripheral and hepatic insulin sensitivity. Non-oxidative glucose disposal and metabolic flexibility (insulin-stimulated respiratory quotient [RQ] minus fasting RQ) were also assessed. Glucose incremental area under the curve was calculated from the OGTT (iAUCOGTT). Exercise increased clamp-derived peripheral and hepatic insulin sensitivity more in adults with IFG or IGT alone than IFG+IGT (P<0.05). Exercise reduced glucose iAUCOGTT in IGT only (P<0.05), and the decrease in glucose iAUCOGTT was inversely correlated with the increase in peripheral, but not hepatic, insulin sensitivity (P<0.01). Increased clamp-derived peripheral insulin sensitivity was also correlated with enhanced metabolic flexibility, reduced fasting RQ, and higher non-oxidative glucose disposal (P<0.05). Adults with IFG+IGT had smaller gains in clamp-derived peripheral insulin sensitivity and metabolic flexibility, which was related to blunted improvements in post-prandial glucose. Further work is required to assess the molecular mechanism(s) by which chronic hyperglycemia modifies insulin sensitivity following exercise training.

    September 24, 2013   doi: 10.1152/ajpendo.00441.2013   open full text
  • Defective prolactin signaling impairs pancreatic beta cell development during the perinatal period.
    Auffret, J., Freemark, M. S., Carre, N., Mathieu, Y., Tourrel-cuzin, C., Lombes, M., Movassat, J., Binart, N.
    AJP Endocrinology and Metabolism. September 24, 2013

    Prolactin (PRL) and placental lactogens stimulate beta cell replication and insulin production in pancreatic islets and insulinoma cells through binding to the PRL receptor (PRLR). However, the contribution of PRLR signaling to beta cell ontogeny and function in perinatal life and the effects of the lactogens on adaptive islet growth are poorly understood. We provide evidence that expansion of beta cell mass during both embryogenesis and the postnatal period is impaired in the PRLR-/- mouse model. PRLR-/- newborns display a 30% reduction of beta cell mass, consistent with reduced proliferation index at E18.5. PRL stimulates leucine incorporation and S6 kinase phosphorylation in INS-1 cells, supporting a role for beta cell mTOR signaling in PRL action. Interestingly, a defect in the development of acini is also observed in absence of PRLR signaling, with a sharp decline in cellular size in both endocrine and exocrine compartments. Of note, a decrease in levels of IGF-2, a PRL target, in the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes, is associated with lack of PRL-mediated beta cell proliferation in embryonic pancreatic buds. Reduced pancreatic IGF2 expression in both rat and mouse models suggests that this factor may constitute a molecular link between PRL signaling and cell ontogenesis. Together, these results provide evidence that PRL signaling is essential for pancreas ontogenesis during the critical perinatal window responsible for establishing functional beta cell reserve.

    September 24, 2013   doi: 10.1152/ajpendo.00636.2012   open full text
  • The rate of production of uric acid by hepatocytes is a sensitive index of compromised cell ATP homeostasis.
    Petrie, J. L., Patman, G. L., Sinha, I., Alexander, T. D., Reeves, H. L., Agius, L.
    AJP Endocrinology and Metabolism. September 17, 2013

    Plasma levels of uric acid, the final product of purine degradation in man, are elevated in metabolic syndrome and are strongly associated with insulin resistance and Nonalcoholic fatty liver disease (NAFLD). Hepatic and blood levels of purine metabolites (inosine, hypoxanthine, xanthine) are also altered in pathophysiological states. We optimized a rat hepatocyte model to test the hypothesis that the production of uric acid by hepatocytes is a potential marker of compromised homeostasis of hepatocellular inorganic phosphate (Pi) and/or ATP. Results: The basal rate of uric acid production from endogenous substrates in rat hepatocytes was comparable to that in human liver and was <10% of the maximum rate with saturating concentrations of purine substrates. It was marginally (~20%) decreased by insulin and increased by glucagon but was stimulated >2-fold by substrates (fructose and glycerol) that lower both cell ATP and Pi; and by inhibitors of mitochondrial respiration (complexes I, III, V) that lower ATP but raise cell Pi. Clearance of inosine and its degradation to uric acid, were also inhibited by cell Pi depletion. Analysis of gene expression in NAFLD biopsies showed an association between mRNA expression of GCKR, the glucokinase regulatory protein that is functionally linked to uric acid production, and mRNA expression of the phosphate transporters encoded by SLC17A1/3. Conclusions: Uric acid production by hepatocytes is a very sensitive index of ATP depletion irrespective of whether cell Pi is lowered or raised. This suggests that raised plasma uric acid may be a marker of compromised hepatic ATP homeostasis.

    September 17, 2013   doi: 10.1152/ajpendo.00214.2013   open full text
  • Nutritional manipulations in the perinatal period program adipose tissue in offspring.
    Lukaszewski, M.-A., Eberle, D., Vieau, D., Breton, C.
    AJP Endocrinology and Metabolism. September 17, 2013

    Epidemiological studies initially demonstrated that maternal undernutrition results in low birth weight with increased risk for long-lasting energy balance disorders. Maternal obesity and diabetes associated with high birth weight, excessive nutrition in neonates and rapid catch-up growth also increase the risk of adult-onset obesity. As stated by the Developmental Origin of Health and Disease concept, nutrient supply perturbations in the fetus or neonate result in long-term programming of individual body weight set-point. Adipose tissue is a key fuel storage unit mainly involved in the maintenance of energy homeostasis. Studies in numerous animal models have demonstrated that the adipose tissue is the focus of developmental programming events in a gender- and depot-specific manner. In rodents, adipose tissue development is particularly active during the perinatal period, especially during the last week of gestation and during early postnatal life. In contrast to rodents, this process essentially takes place before birth in bigger mammals. Despite these different developmental time windows, altricial and precocial species share several mechanisms of adipose tissue programming. Offspring from malnourished dams present adipose tissue with series of alterations: impaired glucose uptake, insulin and leptin resistance, low-grade inflammation, modified sympathetic activity with reduced noradrenergic innervations and thermogenesis. These modifications reprogram adipose tissue metabolism by changing fat distribution and composition, and by enhancing adipogenesis predisposing the offspring to fat accumulation. Subtle adipose tissue circadian rhythm changes are also observed. Inappropriate hormone levels, modified tissue sensitivity (especially glucocorticoid system) and epigenetic mechanisms are key factors for adipose tissue programming during the perinatal period.

    September 17, 2013   doi: 10.1152/ajpendo.00231.2013   open full text
  • Pancreatic beta cell function increases in a linear dose-response manner following exercise training in adults with prediabetes.
    Malin, S. K., Solomon, T. P. J., Blaszczak, A., Finnegan, S., Filion, J., Kirwan, J. P.
    AJP Endocrinology and Metabolism. September 17, 2013

    While some studies suggest that a linear dose-response relationship exists between exercise and insulin sensitivity, the exercise dose required to enhance pancreatic beta-cell function is unknown. Thirty-five older, obese adults with prediabetes underwent a progressive 12-week supervised exercise intervention (5d/wk for 60min at ~85% HRmax). Insulin and C-peptide (n=23) responses to an OGTT were used to define the first and second phase disposition index (DI; beta-cell function = glucose-stimulated insulin secretion x clamp-derived insulin sensitivity). Maximum oxygen consumption (VO2max) and body composition (dual-energy x-ray absorptiometry and computed tomography) were also measured before and after the intervention. Exercise dose was computed using VO2-heart rate derived linear-regression equations. Subjects expended 474.5±8.8 kcal/session (2372.5±44.1 kcal/week) during the intervention, and lost ~8% body weight. Exercise increased first and second phase DI (P<0.05), and these changes in DI were linearly related to exercise dose (DIfirst phase: r=0.54; P<0.001 and DIsecond phase: r=0.56, P=0.0005). Enhanced DI was also associated with increased VO2max (DIfirst phase: r=0.36; P=0.04, DIsecond phase: r=0.41, P<0.02), but not lower body fat (DIfirst phase: r=-0.21; P=0.25 and DIsecond phase: r=-0.30, P=0.10) after training. Low baseline DI predicted an increase in DI after the intervention (DIfirst phase: r=-0.37 and DIsecond phase: r=-0.41, each P<0.04). Thus, exercise training plus weight loss increased pancreatic beta cell function in a linear dose-response manner in adults with prediabetes. Our data suggest that higher exercise doses (i.e. >2000 kcal/week) are necessary to enhance beta-cell function in adults with poor insulin secretion capacity.

    September 17, 2013   doi: 10.1152/ajpendo.00260.2013   open full text
  • Aerobic exercise but not resistance exercise reduces intrahepatic lipid content and visceral fat and improves insulin sensitivity in obese adolescent girls.
    Lee, S., Deldin, A. R., White, D., Kim, Y., Libman, I., Rivera-Vega, M., Kuk, J. L., Sandoval, S., Boesch, C., Arslanian, S.
    AJP Endocrinology and Metabolism. September 17, 2013

    It is unclear whether regular exercise alone (without calorie restriction) is a useful strategy to reduce adiposity and obesity-related metabolic risk factors in obese girls. We examined the effects of aerobic (AE) versus resistance exercise (RE) alone on visceral adipose tissue (VAT), intrahepatic lipid and insulin sensitivity in obese girls. Forty-four obese adolescent girls (BMI >95th, 12-18 yrs) were randomized to 3-months of 180 min/week AE (n=16) or RE (n=16) or a non-exercising control group (n=12). Total fat and VAT were assessed by MRI and intrahepatic lipid by proton magnetic resonance spectroscopy. Intermuscular AT (IMAT) was measured by CT. Insulin sensitivity was evaluated by a 3-hour hyperinsulinemic (80 mU/m2/min)-euglycemic clamp. Compared with controls (0.13 ± 1.10 kg), Body weight did not change (P>0.1) in the AE (-1.31 ± 1.43 kg) and RE (-0.31 ± 1.38 kg) groups. Despite the absence of weight loss, total body fat (%) and IMAT decreased (P<0.05) in both exercise groups compared with control. Compared with control, significant (P<0.05) reductions in VAT ( -15.68 ± 7.64 cm2) and intrahepatic lipid ( -1.70 ± 0.74%), and improvement in insulin sensitivity ( 0.92 ± 0.27 mg/kg/min per µU/ml) were observed in the AE group, but not the RE group. Improvements in insulin sensitivity in the AE group were associated with the reductions in total AT mass (r = -0.65, P=0.02). In obese adolescent girls, aerobic exercise, but not resistance exercise is effective in reducing liver fat, visceral adiposity and improving insulin sensitivity independent of weight loss or calorie restriction.

    September 17, 2013   doi: 10.1152/ajpendo.00285.2013   open full text
  • The dual face of parathyroid hormone and prostaglandins in the osteoimmune system.
    Agas, D., Marchetti, L., Capitani, M., Sabbieti, M. G.
    AJP Endocrinology and Metabolism. September 17, 2013

    The microenvironment of bone marrow, an extraordinarily heterogeneous and dynamic system, is populating by bone and immune cells, and its functional dimension has been at the forefront of recent studies in the field of osteoimmunology. The interaction of both marrow niches supports self-renewal, differentiation and homing of the hematopoietic stem cells, and provides the essential regulatory molecules for osteoblast and osteoclast homeostasis. Impaired signalling within the niches results in a pathological tableau and enhances disease, including osteoporosis and arthritis, or the rejection of hematopoietic stem cell transplants. Discovering the anabolic players that control these mechanisms has become warranted. In this review, we focus on parathyroid hormone (PTH) and prostaglandins (PGs), potent molecular mediators, both of which carry out a multitude of functions, particularly in bone lining cells and T-cells. These two regulators proved to be promising therapeutic agents when strictly clinical protocols on dose treatments were applied.

    September 17, 2013   doi: 10.1152/ajpendo.00290.2013   open full text
  • Human blood contains both the uncleaved precursor of anti-Mullerian hormone (proAMH) and a complex of the N- and C-terminal peptides (AMHN,C).
    Pankhurst, M. W., McLennan, I. S.
    AJP Endocrinology and Metabolism. September 17, 2013

    AMH in blood is a marker of ovarian status in women and the presence of cryptic testes in babies. Despite this, the molecular form of AMH in blood has not been verified. AMH is synthesized as an inert proprotein precursor (proAMH), which can be cleaved to yield N-terminal (AMHN), and C-terminal (AMHC) fragments, that can complex non-covalently (AMHN,C). Developing males have tenfold more AMH than young adults. We report here, that human blood is a mixture of inactive proAMH and receptor-binding AMHN,C. The AMH in the blood of boys, men and premenopausal women was immunoprecipitated using antibodies to the N- and C-terminal peptides. The precipitated proteins were then analyzed by western blots, using recombinant proteins as markers. The glycosylation status of AMH was verified using deglycosylating enzymes. The N-terminal antibody precipitated a major protein that migrated alongside rhproAMH and was detected by anti-AMHN and anti-AMHC. This antibody also precipitated significant levels of AMHN and AMHC from all participants. Antibodies specific to AMHC precipitated rhAMHC, but did not precipitate AMHC from human blood. Hence, all the AMHC in human blood appears to be bound to AMHN. Both AMHN and proAMH were glycosylated, independent of age and sex. In conclusion, boys and young adults have the same form of AMH, with a significant proportion being the inactive precursor. This raises the possibility that the endocrine functions of AMH are partly controlled by its cleavage in the target organ. The presence of proAMH in blood may confound the use of AMH for diagnosis.

    September 17, 2013   doi: 10.1152/ajpendo.00395.2013   open full text
  • Preferential Impact of Pregnancy Associated Plasma Protein-A Deficiency on Visceral Fat in Mice on High Fat Diet.
    Conover, C. A., Harstad, S. L., Tchkonia, T., Kirkland, J. L.
    AJP Endocrinology and Metabolism. September 17, 2013

    Accumulation of visceral fat, more so than subcutaneous fat, is strongly associated with severe metabolic complications. However, the factors regulating depot-specific adipogenesis are poorly understood. In this study, we show differential expression of pregnancy-associated plasma protein-A (PAPP-A), a secreted regulator of local insulin-like growth factor (IGF) action, in adipose tissue of mice. PAPP-A mRNA expression was 5-fold higher in visceral (mesenteric) fat compared to subcutaneous (inguinal, subscapular), peri-renal, and brown fat of mice. To investigate the possible role of depot-specific PAPP-A expression in fat accumulation, wild-type (WT) and PAPP-A knock-out (KO) mice were fed a high-fat diet (HFD) for up to 20 weeks. Adipocyte size increased in subcutaneous and peri-renal depots similarly in WT and PAPP-A KO mice. However, fat cell size and in vivo lipid uptake were significantly reduced in mesenteric fat of PAPP-A KO compared to WT mice. After 20 weeks on HFD, phosphorylation of AKT, a downstream signaling intermediate of IGF-I and insulin receptor activation, was significantly decreased by 50% in mesenteric compared to subcutaneous fat in WT mice, but was significantly increased 3-fold in mesenteric compared to subcutaneous fat in PAPP-A KO mice. This appeared to be due to enhanced insulin-stimulated signaling in mesenteric fat of PAPP-A KO mice. These data establish fat depot-specific expression of PAPP-A, and indicate preferential impact of PAPP-A deficiency on visceral fat in the mouse that is associated with enhanced insulin receptor signaling. Thus, PAPP-A may be a potential target for treatment and/or prevention strategies for visceral obesity and related morbidities.

    September 17, 2013   doi: 10.1152/ajpendo.00405.2013   open full text
  • Palmitoleic acid (n-7) increases white adipocyte lipolysis and lipases content in a PPAR{alpha} dependent manner.
    Bolsoni-Lopes, A., Festuccia, W. T., Farias, T. M., Chimin, P., Torres-Leal, F. L., Derogis, P. B. M., de Andrade, P. B., Miyamoto, S., Lima, F. B., Curi, R., Alonso-Vale, M. I. C.
    AJP Endocrinology and Metabolism. September 10, 2013

    Here we investigated whether palmitoleic acid, a fatty acid that enhances whole body glucose disposal and suppresses hepatic steatosis, modulates triacylglycerol (TAG) metabolism in adipocytes. For this, both differentiated 3T3-L1 cells treated with either palmitoleic acid (16:1n7, 200 µM) or palmitic acid (16:0, 200 µM) for 24h and primary adipocytes from wild type or PPARα deficient mice treated with 16:1n7 (300 mg/kg/day) or oleic acid (18:1n9, 300 mg/kg/day) by gavage for 10 days were evaluated for lipolysis, TAG and glycerol 3-phosphate synthesis and gene and protein expression profile. Treatment of differentiated 3T3-L1 cells with 16:1n7, but not 16:0, increased basal and isoproterenol-stimulated lipolysis, mRNA levels of adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) and protein content of ATGL and pSer660-HSL. Such increase in lipolysis induced by 16:1n7, which can be prevented by pharmacological inhibition of PPARα, was associated with higher rates of PPARα binding to DNA. In contrast to lipolysis, both 16:1n7 and 16:0 increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose, without affecting glyceroneogenesis and glycerokinase expression. Corroborating in vitro findings, treatment of wild type, but not PPARα deficient mice with 16:1n7 increased primary adipocytes basal and stimulated lipolysis and ATGL and HSL mRNA levels. In contrast to lipolysis, however, 16:1n7 treatment increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose in both wild type and PPARα deficient mice. In conclusion, palmitoleic acid increases adipocyte lipolysis and lipases by a mechanism that requires a functional PPARα.

    September 10, 2013   doi: 10.1152/ajpendo.00082.2013   open full text
  • Altered content of AMP-activated protein kinase isoforms in skeletal muscle from spinal cord injured subjects.
    Kostovski, E., Boon, H., Hjeltnes, N., Lundell, L. S., Ahlsen, M., Chibalin, A. V., Krook, A., Iversen, P. O., Widegren, U.
    AJP Endocrinology and Metabolism. September 10, 2013

    AMP-activated protein kinase (AMPK) is a pivotal regulator of energy homeostasis. Although down-stream targets of AMPK are widely characterized, the physiological factors governing isoform expression of this protein kinase are largely unknown. Nerve/contractile activity have a major impact on the metabolic phenotype of skeletal muscle, therefore likely to influence AMPK isoform expression. Spinal cord injury represents an extreme form of physical inactivity, with concomitant changes in skeletal muscle metabolism. We assessed the influence of longstanding and recent spinal cord injury on protein abundance of AMPK isoforms in human skeletal muscle. We also determined muscle fiber type as a marker of glycolytic or oxidative metabolism. In subjects with longstanding complete injury, protein abundance of the AMPK 3 subunit, as well as myosin heavy chain (MHC) IIa and IIx, were increased, whereas abundance of the AMPK 1 subunit and MHC I were decreased. Similarly, abundance of AMPK 3 and MHC IIa proteins was increased, whereas AMPK α2, β1, 1 subunits and MHC I was decreased during the first year following injury, reflecting a more glycolytic phenotype of the skeletal muscle. However, in incomplete cervical lesions, partial recovery of muscle function attenuated the changes in the isoform profile of AMPK and MHC. In conclusion, skeletal muscle abundance of AMPK isoforms is related to physical activity and/or muscle fiber type. Thus, physical/neuromuscular activity is an important determinant of isoform abundance of AMPK and MCH. This further underscores the need for physical activity as part of a treatment regimen to maintain skeletal muscle metabolism.

    September 10, 2013   doi: 10.1152/ajpendo.00132.2013   open full text
  • 6-Mercaptopurine Augments Glucose Transport Activity in Skeletal Muscle Cells in Part via a Mechanism Dependent upon Orphan Nuclear Receptor NR4A3.
    Liu, Q., Zhu, X., Xu, L., Fu, Y., Garvey, W. T.
    AJP Endocrinology and Metabolism. September 10, 2013

    The purine anti-metabolite 6-mercaptopurine (6-MP) is widely used for the treatment of leukemia and inflammatory diseases. The cellular effects of 6-MP on metabolism remain unknown; however, 6-MP was recently found to activate the orphan nuclear receptor NR4A3 in skeletal muscle cell lines. We have previously reported that NR4A3 (also known as NOR-1, MINOR) is a positive regulator of insulin sensitivity in adipocytes. To further explore the role of NR4A3 activation in insulin action we explored whether 6-MP activation of NR4A3 could modulate glucose transport system activity in L6 skeletal muscle cells. We found that 6-MP increased both NR4A3 expression and NR4A3 transcriptional activity, and enhanced glucose transport activity via increasing GLUT4 translocation in both basal and insulin-stimulated L6 cells in an NR4A3 dependent manner. Furthermore, 6-MP increased levels of phospho-AS160 although this effect was not modulated by NR4A3 overexpression or knockdown. These primary findings provide a novel proof of principle that 6-MP, a small molecule NR4A3 agonist, can augment glucose uptake in insulin target cells, although this occurs via both NR4A3 dependent and independent actions; the latter is related to an increase in phospho-AS160. These results establish a novel target for development of new treatments for insulin resistance.

    September 10, 2013   doi: 10.1152/ajpendo.00169.2013   open full text
  • Prolactin Regulates TRPV1, TRPA1 and TRPM8 in Sensory Neurons in Sex-dependent Manner: Contribution of Prolactin Receptor to Inflammatory Pain.
    Patil, M. J., Ruparel, S. B., Henry, M. A., Akopian, A. N.
    AJP Endocrinology and Metabolism. September 10, 2013

    Prolactin (PRL) is a hormone produced in the anterior pituitary but also synthesized extra-pituitary where it can influence diverse cellular processes including inflammatory responses. Females experience greater pain in certain inflammatory conditions, but the contribution of the PRL system to sex-dependent inflammatory pain is unknown. We found that PRL regulates TRP channels in sex-dependent manner in sensory neurons. At >20ng/ml, PRL sensitizes TRPV1 in female, but not male neurons. This effect is mediated by PRL receptor (PRL-R). Likewise, TRPA1 and TRPM8 were sensitized by 100ng/ml PRL only in female neurons. We showed that complete Freund adjuvant (CFA) up-regulated PRL levels in the inflamed paw of both male and female rats, but levels were higher in females. In contrast, CFA did not change mRNA levels of long and short PRL-R in the dorsal root ganglion or spinal cord. Analysis of PRL and PRL-R KO mice demonstrated that basal responses to cold stimuli were only altered in females, and with no significant effects on heat and mechanical responses in both sexes. CFA-induced heat and cold hyperalgesia were not changed in PRL and PRL-R KO compared to WT males, while significant reduction of heat and cold post-CFA hyperalgesia was detected in PRL and PRL-R KO females. Attenuation of CFA-induced mechanical allodynia was observed in both PRL and PRL-R KO females and males. Thermal hyperalgesia in PRL KO females was restored by administration of PRL into hindpaws. Overall, we demonstrate a sex-dependent regulation of peripheral inflammatory hyperalgesia by the PRL system.

    September 10, 2013   doi: 10.1152/ajpendo.00187.2013   open full text
  • Dehydroepiandrosterone exerts anti-glucocorticoid action on human preadipocyte proliferation, differentiation and glucose uptake.
    McNelis, J. C., Manolopoulos, K. N., Gathercole, L. L., Bujalska, I. J., Stewart, P. M., Tomlinson, J. W., Arlt, W.
    AJP Endocrinology and Metabolism. September 10, 2013

    Glucocorticoids increase adipocyte proliferation and differentiation, a process underpinned by the local reactivation of inactive cortisone to active cortisol within adipocytes, catalyzed by 11β-hydroxysteroid dehydrogenase type (11β-HSD1). The adrenal sex steroid precursor dehydroepiandrosterone (DHEA) has been shown to inhibit 11β-HSD1 in murine adipocytes; however, rodent adrenals do not physiologically produce DHEA. Here we aimed to determine the effects and underlying mechanisms of the potential anti-glucocorticoid action of DHEA and its sulfate ester DHEAS in human preadipocytes. Utilizing a human subcutaneous preadipocyte cell line, Chub-S7, we examined the metabolism and effects of DHEA in human adipocytes, including adipocyte proliferation, differentiation, 11β-HSD1 expression and activity and glucose uptake. DHEA, but not DHEAS, significantly inhibited preadipocyte proliferation via cell cycle arrest in G1 phase, independent of sex steroid and glucocorticoid receptor activation. 11β-HSD1 oxoreductase activity in differentiated adipocytes was inhibited by DHEA. DHEA co-incubated with cortisone significantly inhibited preadipocyte differentiation, assessed by the expression of markers of early (LPL) and terminal (G3PDH) adipocyte differentiation. Co-incubation with cortisol, negating the requirement for 11β-HSD1 oxoreductase activity, diminished the inhibitory effect of DHEA. Further consistent with glucocorticoid-opposing effects of DHEA, insulin-independent glucose uptake was significantly enhanced by DHEA treatment. DHEA increases basal glucose uptake and inhibits human preadipocyte proliferation and differentiation, thereby exerting an anti-glucocorticoid action. DHEA inhibition of the amplification of glucocorticoid action mediated by 11β-HSD1 contributes to the inhibitory effect of DHEA on human preadipocyte differentiation.

    September 10, 2013   doi: 10.1152/ajpendo.00314.2012   open full text
  • The eukaryotic initiation factor 2 (eIF2) kinase GCN2 protects against hepatotoxicity during asparaginase treatment.
    Wilson, G. J., Bunpo, P., Cundiff, J. K., Wek, R. C., Anthony, T. G.
    AJP Endocrinology and Metabolism. September 03, 2013

    Asparaginase is an important drug in the treatment regimen for acute lymphoblastic leukemia. Asparaginase depletes circulating asparagine and glutamine, activating an amino acid stress response (AAR) involving phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible kinase 2 (GCN2). We hypothesized that GCN2 functions to mitigate hepatic stress during asparaginase therapy by activating the AAR. To test this idea, C57BL/6J wild-type mice (GCN2+/+) and those deleted for GCN2 (GCN2-/-) were injected with asparaginase or saline excipient once daily for 1 or 6 d. In liver, increased phosphorylation of eIF2 and mRNA expression of AAR target genes ATF4, ASNS, 4E-BP1, and CHOP were significantly blunted or blocked in the liver of GCN2-/- mice. Loss of AAR during asparaginase coincided with increases in mammalian target of rapamycin (mTOR) signaling, hepatic triglyceride accumulation, and DNA damage in association with markers of oxidative stress (GPX1) and inflammation (TNFalpha). While asparaginase depleted circulating asparagine in both GCN2+/+ and GCN2-/- mice, all other amino acids including plasma glutamine were elevated in the plasma of GCN2-/- mice.This study shows that loss of GCN2 promotes oxidative stress and inflammatory-mediated DNA damage during asparaginase therapy, suggesting that patients with reduced or dysfunctional AAR may be at risk of developing hepatic complications during asparaginase treatment.

    September 03, 2013   doi: 10.1152/ajpendo.00080.2013   open full text
  • Mouse and Human Islets Survive and Function After Coating by Biosilicification.
    Jaroch, D. B., Lu, J., Madangopal, R., Stull, N. D., Stensberg, M., Shi, J., Kahn, J. L., Herrera-Perez, R., Zeitchek, M., Sturgis, J., Robinson, J. P., Yoder, M. C., Porterfield, D. M., Mirmira, R. G., Rickus, J. L.
    AJP Endocrinology and Metabolism. September 03, 2013

    Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic / soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immuno-protection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to media containing saturating silicic acid levels for 9 to 15 minutes. The resulting tissue constructs were then cultured for up to four weeks under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. Cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function was assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice.

    September 03, 2013   doi: 10.1152/ajpendo.00081.2013   open full text
  • Alpha-cells are dispensable in postnatal morphogenesis and maturation of mouse pancreatic islets.
    Shiota, C., Prasadan, K., Guo, P., El-Gohary, Y., Wiersch, J., Xiao, X., Esni, F., Gittes, G. K.
    AJP Endocrinology and Metabolism. August 27, 2013

    Glucagon-producing α-cells are the second most abundant cell type in the islet. While α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphologic and functional maturation occurs to form adult islets. To determine if α-cells have a role in postnatal islet development, a diphtheria toxin mediated α-cell ablation mouse model was established. Rapid and persistant depletion of α-cells was achieved by daily injection of the toxin for 2 weeks starting at post-natal day 1 (P1). Total pancreatic glucagon content in the α-cell ablated mice was undetectable at P14, and still less than 0.3% of that of the control mice at 4 months of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near total lack of α-cells. Furthermore, there were no differences in expression of β-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis nor functional maturation of β-cells.

    August 27, 2013   doi: 10.1152/ajpendo.00022.2013   open full text
  • Altered Subcutaneous Abdominal Adipose Tissue Lipid Synthesis in Obese, Insulin Resistant Humans.
    Tuvdendorj, D., Chandalia, M., Batbayar, T., Saraf, M., Beysen, C., Murphy, E. J., Abate, N.
    AJP Endocrinology and Metabolism. August 27, 2013

    The purpose of this study was to evaluate the variability of subcutaneous abdominal adipose tissue (AT) dynamics in obese subjects with a wide range of insulin sensitivity (IS) and the correlation between these two metabolic measures IS. Ten obese (BMI 30-40 kg/m2) non-diabetic subjects with (n=6) and without (n=4) the metabolic syndrome were studied following a 12 week 2H2O labeling period. Subcutaneous abdominal AT biopsies were collected. Deuterium incorporation into TG-glycerol and TG-palmitate were measured by gas-chromatography-mass-spectrometry for the calculation of fractional TG synthesis (fTG) and fractional de novo lipogenesis (fDNL). Muscle IS and insulin-mediated non-esterified fatty acid (NEFA) suppression (a measure for adipose IS) indices were derived from the oral glucose tolerance test (OGTT). The ability of subcutaneous abdominal AT to synthesize lipids varied significantly in obese subjects (fTG range 7-28% and fDNL range 1.1-4.6%) with significantly lower values (>35% reduction) for both parameters in obese with the metabolic syndrome. fTG correlated positively with muscle IS (r=0.64, P=0.04) and inversely with NEFA suppression during the OGTT (r=-0.69, P=0.03). These results demonstrate a large variability in subcutaneous abdominal AT lipid turnover in obesity. Moreover, a reduced capacity for subcutaneous abdominal AT fat storage is associated with muscle and adipose tissue insulin resistance, as well as with the metabolic syndrome and, thus identifying a form of obesity at heightened risk for type 2 diabetes and cardiovascular disease.

    August 27, 2013   doi: 10.1152/ajpendo.00194.2013   open full text
  • Impaired glucose tolerance in rats fed low-carbohydrate, high-fat diets.
    Bielohuby, M., Sisley, S., Sandoval, D. A., Herbach, N., Zengin, A., Fischereder, M., Menhofer, D., Stoehr, B. J. M., Stemmer, K., Wanke, R., Tschop, M. H., Seeley, R. J., Bidlingmaier, M.
    AJP Endocrinology and Metabolism. August 27, 2013

    Moderate low-carbohydrate/high-fat diets (LC-HFDs) are widely used to induce weight loss in overweight subjects, whereas extreme ketogenic LC-HFDs are used to treat neurological disorders like pediatric epilepsy. Usage of LC-HFD for improvement of glucose metabolism is highly controversial: some studies suggest that LC-HFDs ameliorate glucose tolerance whereas others could not find positive effects of these diets or reported impaired insulin sensitivity. Here we investigate the effects of LC-HFDs on glucose and insulin metabolism in a well-characterised animal model: rats were fed isoenergetic or hypocaloric amounts of standard control diet, a high-protein, "Atkins-style" LC-HFD, or a low-protein, ketogenic, LC-HFD. Both LC-HFDs induced lower fasting glucose and insulin levels associated with lower pancreatic β-cell volumes. However, dynamic challenge tests (oral and intraperitoneal glucose tolerance tests, insulin-tolerance tests and hyperinsulinemic-euglycemic clamps) revealed that LC-HFD pair-fed rats exhibited impaired glucose tolerance, and impaired hepatic and peripheral tissue insulin sensitivity, the latter potentially being mediated by elevated intramyocellular lipids. Adjusting visceral fat mass in LC-HF groups to that of controls by reducing the intake of LC-HF diets to 80% of the pair-fed groups did not prevent glucose intolerance. Taken together, these data show that lack of dietary carbohydrates leads to glucose intolerance and insulin resistance in rats despite causing a reduction in fasting glucose and insulin concentrations. Our results argue against beneficial effects of LC-HFDs on glucose and insulin metabolism, at least under physiological conditions. Therefore, use of LC-HFDs for weight loss or other therapeutic purposes should be balanced against potentially harmful metabolic side effects.

    August 27, 2013   doi: 10.1152/ajpendo.00208.2013   open full text
  • Mitochondrial and performance adaptations to exercise training in mice lacking skeletal muscle LKB1.
    Tanner, C. B., Madsen, S. R., Hallowell, D. M., Goring, D. M. J., Moore, T. M., Hardman, S. E., Heninger, M. R., Atwood, D. R., Thomson, D. M.
    AJP Endocrinology and Metabolism. August 27, 2013

    LKB1 and its downstream targets of the AMP-activated protein kinase family are important regulators of many aspects of skeletal muscle cell function, including control of mitochondrial content and capillarity. LKB1 deficiency in skeletal and cardiac muscle (mLKB1-KO) greatly impairs exercise capacity. However, cardiac dysfunction in that genetic model prevents a clear assessment of the role of skeletal muscle LKB1 in the observed effects. Our purposes here were to determine whether skeletal muscle specific knockout of LKB1 (skmLKB1-KO) decreases exercise capacity and mitochondrial protein content, impairs accretion of mitochondrial proteins after exercise training, and attenuates improvement in running performance after exercise training. We found that treadmill and voluntary wheel running capacity was reduced in skmLKB1-KO vs. control (CON) mice. Citrate synthase (CS) activity, succinate dehydrogenase activity and pyruvate dehydrogenase kinase content were lower in KO vs. CON muscles. Three weeks of treadmill training resulted in significantly increased treadmill running performance in both CON and skmLKB1-KO mice. CS activity increased significantly with training in both genotypes, but protein content and activity for components of the mitochondrial electron transport chain increased only in CON mice. Capillarity and VEGF protein was lower in skmLKB1-KO vs. CON muscles but VEGF increased with training only in skmLKB1-KO. Three hours after an acute bout of muscle contractions, PGC-1α, cytochrome C, and VEGF gene expression all increased in CON but not skmLKB1-KO muscles. Our findings indicate that skeletal muscle LKB1 is required for accretion of some mitochondrial proteins, but not for early exercise capacity improvements with exercise training.

    August 27, 2013   doi: 10.1152/ajpendo.00227.2013   open full text
  • The insulin receptor plays an important role in secretory differentiation in the mammary gland.
    Neville, M. C., Webb, P., Ramanathan, P., Manninno, M. P., Pecorini, C., Monks, J., Anderson, S. M., MacLean, P. S.
    AJP Endocrinology and Metabolism. August 27, 2013

    Insulin is known to be an important regulator of milk secretion in the lactating mammary gland. Here we examine the role of insulin signaling in mammary development in pregnancy using a mouse with a floxed insulin receptor (IR) crossed with a mouse expressing Cre specifically in the mammary gland. In the mammary glands of these IRfl/fl Cre+ mice expression of IR is significantly diminished throughout development. Glands from these mice had 50% fewer alveoli at mid-pregnancy; casein and lipid droplets were diminished by 60% and 75% respectively, indicating a role for IR both in alveolar development and differentiation. In an acinar preparation from mammary epithelial cells (MEC) isolated from pregnant mice, insulin stimulated lumen formation, mammary cell size, acinar size, acinar casein content and the formation of lipid droplets with a Km of ~1.7 nM. IGF1 and IGF2 had no effect at concentrations below 50 nM and a function blocking antibody to the IGF type 1 receptor did not alter the response to insulin. We conclude that insulin interacting with IR is essential for mammary differentiation during murine pregnancy. Using array analysis we then examined the expression of genes up- or down-regulated >1.5 fold in the IRfl/fl Cre+ mammary epithelial cells finding significant down-regulation of differentiation specific genes and up-regulation of cell cycle and extracellular matrix genes. We conclude that insulin fosters differentiation and may inhibit cell proliferation in the mammary gland of the mid-pregnant mouse.

    August 27, 2013   doi: 10.1152/ajpendo.00337.2013   open full text
  • Intracellular lipid content is a key intrinsic determinant for hepatocyte viability and metabolic and inflammatory states in mice.
    Sheng, L., Jiang, B., Rui, L.
    AJP Endocrinology and Metabolism. August 27, 2013

    The liver is an essential metabolic organ. In addition to metabolizing glucose and lipids, hepatocytes also secrete various cytokines which modulate both hepatocyte metabolism and liver inflammation. Hepatocyte injury and death and liver inflammation are the major contributors to liver diseases, including nonalcoholic steatohepatitis (NASH). Anatomic locations have a profound effect on hepatocyte metabolism, and liver zonation describes the metabolic heterogeneity of hepatocytes along the portovenous axis. However, it is unclear whether hepatocyte heterogeneity is affected by intrinsic factors and whether dietary fat, a risk factor for NASH, has distinct detrimental effects on different hepatocyte subpopulations. Here we showed that mouse livers contained both high-lipid and low-lipid subpopulations of hepatocytes. The high-lipid subpopulation was more susceptible to injury and apoptosis, and produced more proinflamatrory cytokines after treatment with endotoxin and saturate fatty acids. Dietary fat consumption further increased fatty acid uptake, intracellular lipid levels, hepatocyte injury and death, and the expression of proinflammatory cytokines in the high-lipid subpopulation. In contrast, dietary fat slightly increased lipid levels, cell death, and expression of proinflammatory cytokines in the low-lipid subpopulation. The low-lipid subpopulation produced more glucose. Fat consumption further activated the gluconeogenic program in the low-lipid, but not the high-lipid, subpopulations. These data suggest that intracellular lipid content is a key intrinsic determinant for hepatocyte heterogeneity of metabolic, inflammatory, and survival states.

    August 27, 2013   doi: 10.1152/ajpendo.00401.2013   open full text
  • Metformin prevents liver tumorigenesis induced by high-fat diet in C57Bl/6 Mice.
    Tajima, K., Nakamura, A., Shirakawa, J., Togashi, Y., Orime, K., Sato, K., Inoue, H., Kaji, M., Sakamoto, E., Ito, Y., Aoki, K., Nagashima, Y., Atsumi, T., Terauchi, Y.
    AJP Endocrinology and Metabolism. August 20, 2013

    The prevalence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is increasing with the growing epidemics of obesity and diabetes. NAFLD encompasses a clinicopathologic spectrum of disease ranging from isolated hepatic steatosis to NASH, which is a more aggressive form of fatty liver disease, to cirrhosis and finally, hepatocellular carcinoma (HCC). The exact mechanism behind the development of HCC in NASH remains unclear; however it has been established that hepatic steatosis is the important risk factor in the development of HCC. Metformin has recently drawn attention because of its potential antitumor effect. Here, we investigated the effects of metformin on high-fat diet (HFD)-induced liver tumorigenesis using a mouse model of NASH and liver tumor. Metformin prevented long-term HFD-induced liver tumorigenesis in C57Bl/6 mice. Of note, metformin failed to protect against liver tumorigenesis in mice that had already begun to develop NAFLD. Metformin improved short-term HFD-induced fat accumulation in the liver, associated with the suppression of adipose tissue inflammation. Collectively, these results suggest that metformin may prevent liver tumorigenesis via suppression of liver fat accumulation in the early stage, before the onset of NAFLD, which seems to be associated with a delay in the development of inflammation of the adipose tissue.

    August 20, 2013   doi: 10.1152/ajpendo.00133.2013   open full text
  • QRFP induces aldosterone production via PKC and T-type calcium channel mediated pathways in human adrenocortical cells: evidence for a novel role of GPR103.
    Ramanjaneya, M., Karteris, E., Chen, J., Rucinski, M., Ziolkowska, A., Ahmed, N., Kagerer, S., Johren, O., Lehnert, H., Malendowicz, L. K., Randeva, H. S.
    AJP Endocrinology and Metabolism. August 20, 2013

    Hormonal regulation of adrenal function occurs primarily through activation of GPCRs. GPCRs are central to many of the body's endocrine and neurotransmitter pathways. Recently it was shown that activation of GPR103 by its ligand QRFP induced feeding, locomotor activity and metabolic rate, and QRFP is bioactive in adipose tissue of obese individuals. Given that the adrenal gland is pivotal organ for energy balance and homeostasis we hypothesised that GPR103 and QRFP are involved in steroidogenic responses. Using qRT-PCR and immunohistochemistry we mapped both GPR103 and QRFP in human fetal and adult adrenal gland as well as rat adrenals. Both were primarily localised in the adrenal cortex but not in the medulla. Activation of GPR103 in human adrenocortical H295R cells led to a decrease in forskolin-increased cAMP and increase of intracellular Ca++ levels. In addition, treatment of H295R cells with QRFP induced aldosterone and cortisol secretion as measured by ELISA. These increases were accompanied by increased expression and activity of StAR, CYB11B1 and CYP11B2 as assessed by qRT-PCR and luciferase-reporter assay respectively. Using specific inhibitors we also demonstrated that the aldosterone induction involves MAPK, PKC and/or T-type Ca++ channel dependent pathways. These novel data demonstrate that QRFP induces adrenal steroidogenesis in vitro by regulating key steroidogenic enzymes involving MAPK/PKC and Ca++ signalling pathways.

    August 20, 2013   doi: 10.1152/ajpendo.00191.2013   open full text
  • Higher activation of autophagy in skeletal muscle of mice during endurance exercise in the fasted state.
    Jamart, C., Naslain, D., Gilson, H., Francaux, M.
    AJP Endocrinology and Metabolism. August 20, 2013

    Activation of autophagy in skeletal muscle has been reported in response to endurance exercise and food deprivation, independently. The purpose of this study was to evaluate whether autophagy was more activated when both stimuli were combined, namely when endurance exercise was performed in a fasted rather than a fed state. Mice performed a low intensity running exercise (10m/min for 90min) in both dietary states after which the gastrocnemius muscles were removed. LC3b-II, a marker of autophagosome presence, increased in both conditions, but the increase was higher in the fasted state. Other protein markers of autophagy like Gabarapl1-II and Atg12 conjugated form as well as mRNA of Lc3b, Gabarapl1 and p62/Sqstm1 were only increased when exercise was performed in a fasted state. The larger activation of autophagy by exercise in a fasted state was associated with a larger decrease in plasma insulin and phosphorylation of AktSer473, AktThr308, FoxO3aThr32 and ULK1Ser757. AMPKαThr172, ULK1Ser317 and ULK1Ser555 remained unchanged in both conditions whereas p38Thr180Tyr182 increased during exercise to a similar extent in the fasted and fed conditions. The marker of mitochondrial fission DRP1Ser616 was increased by exercise, independently of the nutritional status. Changes in mitophagy markers BNIP3 and Parkin suggest that mitophagy was increased during exercise in the fasted state. In conclusion, our results highlight a major implication of the insulin/Akt/mTOR pathway and its downstream targets FoxO3a and ULK1 in the larger activation of autophagy observed when exercise is performed in a fasted state, as compared to a fed state.

    August 20, 2013   doi: 10.1152/ajpendo.00270.2013   open full text
  • Loss of CCR5 results in glucose intolerance in diet-induced obese mice.
    Kennedy, A. J., Webb, C. D., Hill, A. A., Gruen, M. L., Jackson, L. G., Hasty, A. H.
    AJP Endocrinology and Metabolism. August 13, 2013

    Macrophage and T cell infiltration into metabolic tissues contributes to obesity-associated inflammation and insulin resistance (IR). C-C Chemokine Receptor 5 (CCR5), expressed on macrophages and T cells, plays a critical role in the recruitment and activation of pro-inflammatory M1 and TH1 immune cells to tissues and is elevated in adipose tissue (AT) and liver of obese humans and mice. Thus, we hypothesized that deficiency of CCR5 would protect against diet-induced inflammation and IR. CCR5 deficient (CCR5-/-) and C57BL/6 (WT) controls were fed 10% low fat (LF) or 60% high fat (HF) diets for 16 weeks. HF feeding increased adiposity, blood glucose, and plasma insulin levels equally in both genotypes. Opposing our hypothesis, HF-fed CCR5-/- mice were significantly more glucose intolerant than WT mice. In AT, there was a significant reduction in the M1 associated gene, CD11c, while M2 associated genes were not different between genotypes. In addition, HF feeding caused a 2-fold increase in CD4+ T cells in the AT of CCR5-/- compared to WT mice. In liver and muscle, no differences in immune cell infiltration or inflammatory cytokine expression were detected. However in AT and muscle, there was a mild reduction in insulin-induced phosphorylation of AKT and IRβ in CCR5-/- compared to WT mice. These findings suggest that while CCR5 plays a minor role in regulating immune cell infiltration and inflammation in metabolic tissues, deficiency of CCR5 impairs systemic glucose tolerance as well as AT and muscle insulin signaling.

    August 13, 2013   doi: 10.1152/ajpendo.00177.2013   open full text
  • Caveolin-1 upregulation in diabetic fibroblasts and wounded tissues: implication for understanding the underlying mechanisms of non-healing diabetic ulcers.
    Bitar, M. S., Abdel-Halim, S. M., Al-Mulla, F.
    AJP Endocrinology and Metabolism. August 13, 2013

    A heightened state of oxidative stress and senescence of fibroblasts constitute potential therapeutic targets in non-healing diabetic wounds. Here, we studied the underlying mechanism mediating diabetes-induced cellular senescence using in vitro cultured dermal fibroblasts and in vivo circular wounds. Our results demonstrated that the total antioxidant capacity, mRNA levels of thioredoxinreductase and glucose-6-phosphate dehydrogenase as well as the ratio of NADPH/NADP were markedly decreased in fibroblasts from patients with type 2 diabetes (DFs). Consistent with this shifts in favor of excessive reactive oxygen species, DFs also displayed a significant increase in senescence-associated β-galactosidase activity and phospho--histone H2AX (pH2AX) level. Moreover, the ability of PDGF to promote cell proliferation/migration and to regulate the phosphorylation-dependent activation of Akt and ERK1/2 appear to be attenuated as a function of diabetes. Mechanistically, we found that diabetes-induced oxidative stress up-regulated caveolin-1 (Cav-1) and PTRF expression, which in turn sequestered Mdm2 away from p53. This process resulted in the activation of a p53/p21-dependent pathway and the induction of premature senescence in DFs. Most of the aforementioned oxidative stress and senescence-based features observed in DFs were recapitulated in a 10-day-old diabetic wound. Intriguingly, we confirmed that the targeted depletion of Cav-1 or PTRF using siRNA- or Vivo-Morpholino antisense-based gene therapy markedly inhibited diabetes/oxidative stress-induced premature senescence and also accelerated tissue repair in this disease state. Overall, our data illuminate Cav-1/PTRF-1 as a key player of a novel signaling pathway that may link a heightened state of oxidative stress to cellular senescence and impaired wound healing in diabetes

    August 13, 2013   doi: 10.1152/ajpendo.00189.2013   open full text
  • Role of stearoyl-CoA desaturase-1 in skeletal muscle function and metabolism.
    Stamatikos, A. D., Paton, C. M.
    AJP Endocrinology and Metabolism. August 13, 2013

    Stearoyl-CoA desaturase-1 (SCD1) converts saturated fatty acids (SFA) into monounsaturated fatty acids and is necessary for proper liver, adipose tissue, and skeletal muscle lipid metabolism. While there is a wealth of information regarding SCD1 expression in the liver, research on its effect in skeletal muscle is scarce. Furthermore, the majority of information about its role is derived from global knockout mice which are known to be hypermetabolic and fail to accumulate SCD1's substrate, SFA. We now know that SCD1 expression is important in regulating lipid bilayer fluidity, increasing triglyceride formation, enabling lipogenesis, and may protect against SFA-induced lipotoxicity. Exercise has been shown to increase SCD1 expression which may contribute to an increase in intra-myocellular triglyceride at the expense of free fatty acids and diacylglycerol. This review is intended to define the role of SCD1 in skeletal muscle and discuss the potential benefits of its activity in the context of lipid metabolism, insulin sensitivity, exercise training, and obesity.

    August 13, 2013   doi: 10.1152/ajpendo.00268.2013   open full text
  • The Effects of Recovery Sleep after One Workweek of Mild Sleep Restriction on Interleukin-6 and Cortisol Secretion and Daytime Sleepiness and Performance.
    Pejovic, S., Basta, M., Vgontzas, A. N., Kritikou, I., Shaffer, M. L., Tsaoussoglou, M., Stiffler, D., Stefanakis, Z., Bixler, E. O., Chrousos, G. P.
    AJP Endocrinology and Metabolism. August 13, 2013

    One workweek of mild sleep restriction adversely impacts sleepiness, performance and pro-inflammatory cytokines. Many individuals try to overcome these adverse effects by extending their sleep on weekends. To assess whether extended recovery sleep reverses the effects of mild sleep restriction on sleepiness/alertness, inflammation and stress hormones, 30 healthy, young men and women (mean age ±SD, 24.7 ± 3.5; mean body mass index ±SD, 23.6 ± 2.4 kg/m2) participated in a sleep laboratory experiment of 13 nights [4 baseline nights (8h/night), followed by 6 sleep restriction nights (6h/night) and 3 recovery nights (10h /night)]. Twenty-four-hour profiles of circulating interleukin-6 (IL-6) and cortisol, objective and subjective daytime sleepiness (Multiple Sleep Latency Test and Stanford Sleepiness Scale), and performance (Psychomotor Vigilance Task) were assessed on days 4 (baseline), 10 (after one week of sleep restriction) and 13 (after 2 nights of recovery sleep). Serial 24-h IL-6 plasma levels increased significantly during sleep restriction and returned to baseline after recovery sleep. Serial 24-h cortisol levels during restriction did not change compared to baseline, but after recovery they were significantly lower. Subjective and objective sleepiness increased significantly after restriction and returned to baseline after recovery. In contrast, performance deteriorated significantly after restriction and did not improve after recovery. Extended recovery sleep over the weekend reverses the impact of one workweek of mild sleep restriction on daytime sleepiness, fatigue and IL-6 levels, reduces cortisol levels, but does not correct performance deficits. The long-term effects of a repeated sleep restriction/sleep recovery weekly cycle in humans remain unknown.

    August 13, 2013   doi: 10.1152/ajpendo.00301.2013   open full text
  • Direct and Indirect Effects of Growth Hormone Receptor Ablation on Liver Expression of Xenobiotic Metabolizing Genes.
    Li, X., Bartke, A., Berryman, D. E., Funk, K., Kopchick, J. J., List, E. O., Sun, L. Y., Miller, R. A.
    AJP Endocrinology and Metabolism. August 13, 2013

    Detoxification of ingested xenobiotic chemicals, and of potentially toxic endogenous metabolites, is carried out largely through a series of enzymes synthesized in the liver, sometimes called "xenobiotic metabolizing enzymes" (XME). Expression of these XME is sexually dimorphic in rodents and humans, with many of the XME expressed at higher levels in females. This expression pattern is thought to be regulated, in part, by the sex differences in circadian growth hormone (GH) pulsatility. We have evaluated mRNA, in the liver, for 52 XME genes in male and female mice of four mutant stocks, with diminished levels of GH receptor (GHR) either globally (GKO), or in liver (LKO), fat (FKO), or muscle (MKO) tissue specifically. The data show complex, sex-specific changes. For some XME, the expression pattern is consistent with direct control of hepatic mRNA by GHR in the liver. In contrast, other XME show evidence for indirect pathways in which hepatic XME expression is altered by GH signals in fat or skeletal muscle. The effects of GHR-null mutations on glucose control, responses to dietary interventions, steroid metabolism, detoxification pathways and lifespan may depend on a mixture of direct hepatic effects and cross-talk between different GH-responsive tissues.

    August 13, 2013   doi: 10.1152/ajpendo.00304.2013   open full text
  • Arterio-venous balance studies of skeletal muscle fatty acid metabolism - what can we believe?
    Guo, Z., Jensen, M. D.
    AJP Endocrinology and Metabolism. August 13, 2013

    The arterio-venous balance (A-V balance/difference) technique has been used by a number of groups, including ours, to study skeletal muscle fatty acid metabolism. Several lines of evidence indicate that like glycogen, intramyocellular triglycerides (imcTG) are an energy source for local use. As such, the report that increased release of free fatty acids (FFA) via lipolysis from skeletal muscle, but not from adipose tissue, is responsible for the increased systemic lipolysis during IL-6 infusion in healthy humans is somewhat unexpected (26). It appears that given the complex anatomy of human limbs, as to be discussed below, it is virtually impossible to determine whether any fatty acids being released into the venous circulation of an arm or leg derive from the lipolysis of intermuscular fat residing between muscle groups, intramuscular fat residing within muscle groups (between epimysium and perimysium, or bundles), or from the intramyocellular triglyceride droplets (imcTG). In many cases, it may be even difficult to be confident there is no contribution of FFA from subcutaneous adipose tissue. This question is fundamentally important as one attempts to interpret the results of skeletal muscle fatty acid metabolism studies using the A-V balance technique. In this mini review, we examine the reported results of fatty acid kinetics obtained using the techniques to evaluate the degree of and how to minimize contamination when attempting to sample skeletal muscle-specific fatty acids.

    August 13, 2013   doi: 10.1152/ajpendo.00346.2013   open full text
  • Skeletal Muscle Denervation Causes Skeletal Muscle Atrophy Through a Pathway that Involves Both Gadd45a and HDAC4.
    Bongers, K. S., Fox, D. K., Ebert, S. M., Kunkel, S. D., Dyle, M. C., Bullard, S. A., Dierdorff, J. M., Adams, C. M.
    AJP Endocrinology and Metabolism. August 13, 2013

    Skeletal muscle denervation causes muscle atrophy via complex molecular mechanisms that are not well understood. To better understand these mechanisms, we investigated how muscle denervation increases Gadd45a mRNA in skeletal muscle. Previous studies established that muscle denervation strongly induces Gadd45a mRNA, which increases Gadd45a, a small myonuclear protein that is required for denervation-induced muscle fiber atrophy. However, the mechanism by which denervation increases Gadd45a mRNA remained unknown. Here, we demonstrate that histone deacetylase 4 (HDAC4) mediates induction of Gadd45a mRNA in denervated muscle. Using mouse models, we show that HDAC4 is required for induction of Gadd45a mRNA during muscle denervation. Conversely, forced expression of HDAC4 is sufficient to increase skeletal muscle Gadd45a mRNA in the absence of muscle denervation. Moreover, Gadd45a mediates several downstream effects of HDAC4 including induction of myogenin mRNA, induction of mRNAs encoding the embryonic nicotinic acetylcholine receptor, and, most importantly, skeletal muscle fiber atrophy. Because Gadd45a induction is also a key event in fasting-induced muscle atrophy, we tested whether HDAC4 might also contribute to Gadd45a induction during fasting. Interestingly, however, HDAC4 is not required for fasting-induced Gadd45a expression or muscle atrophy. Furthermore, the transcription factor ATF4, which contributes to fasting-induced Gadd45a expression, is not required for denervation-induced Gadd45a expression or muscle atrophy. Collectively, these results identify HDAC4 as an important regulator of Gadd45a in denervation-induced muscle atrophy, and elucidate Gadd45a as a convergence point for distinct upstream regulators during muscle denervation and fasting.

    August 13, 2013   doi: 10.1152/ajpendo.00380.2013   open full text
  • A form of Mitofusin 2 (Mfn2) lacking the transmembrane domains and the C-terminal end stimulates metabolism in muscle and liver cells.
    Segales, J., Paz, J. C., Hernandez-Alvarez, M. I., Sala, D., Munoz, J. P., Noguera, E., Pich, S., Palacin, M., Enriquez, J. A., Zorzano, A.
    AJP Endocrinology and Metabolism. August 13, 2013

    Mitofusin 2 (Mfn2), a protein that participates in mitochondrial fusion, is required to maintain normal mitochondrial metabolism in skeletal muscle and in liver. Given that muscle Mfn2 is repressed in obese or type 2 diabetic subjects, this protein may have a potential pathophysiological role in these conditions. In order to evaluate whether the metabolic effects of Mfn2 can be dissociated from its function in mitochondrial dynamics, we have studied a form of Mfn2, lacking the two transmembrane domains and the C-terminal coiled-coil (Mfn2). This form localized in mitochondria but did not alter mitochondrial morphology in cells or in skeletal muscle fibers. The expression of Mfn2 in mouse skeletal muscle stimulated glucose oxidation and enhanced respiratory control ratio, which occurred in the absence of changes in mitochondrial mass. The expression of Mfn2 in mouse liver or in hepatoma cells stimulated gluconeogenesis. In addition, Mfn2 activated basal and maximal respiration both in muscle and liver cells. In all, we show that a form of Mfn2 lacking mitochondrial fusion activity stimulates mitochondrial function, and enhances glucose metabolism in muscle and liver tissues. This study suggests that Mfn2 regulates metabolism independently of changes in mitochondrial morphology.

    August 13, 2013   doi: 10.1152/ajpendo.00546.2012   open full text
  • Cyclin-dependent kinase 5 modulates STAT3 and androgen receptor activation through phosphorylation of Ser727 on STAT3 in prostate cancer cells.
    Hsu, F.-N., Chen, M.-C., Lin, K.-C., Peng, Y.-T., Li, P.-C., Lin, E., Chiang, M.-C., Hsieh, J.-T., Lin, H.
    AJP Endocrinology and Metabolism. August 13, 2013

    Cyclin-dependent kinase 5 (Cdk5) is known to regulate prostate cancer metastasis. Our previous results indicate that Cdk5 activates androgen receptor (AR) and supports prostate cancer growth. We also find that STAT3 is a target of Cdk5 in promoting thyroid cancer cell growth, while STAT3 may play a regulator to AR activation under cytokine control. This study is to investigate the regulation of Cdk5 and its activator p35 on STAT3/AR signaling in prostate cancer cells. Our results show that Cdk5 biochemically interacts with STAT3 and this interaction depends on Cdk5 activation in prostate cancer cells. The phosphorylation of STAT3 at serine-727 (p-S727-STAT3) is regulated by Cdk5 in cells and xenograft tumors. The mutant of STAT3 S727A reduces its interaction with Cdk5. We further show that the nuclear distribution of p-S727-STAT3 and the expression of STAT3-regulated genes (junB, c-fos, c-myc and survivin) are regulated by Cdk5 activation. STAT3 mutant does not further decrease cell proliferation upon Cdk5 inhibition, which implies that the role of STAT3 regulated by Cdk5 correlates to cell proliferation control. Interestingly, Cdk5 may regulate the interaction between STAT3 and AR through phosphorylation of S727-STAT3 and therefore up-regulate AR protein stability and transactivation. Correspondingly, clinical evidence shows that the level of p-S727-STAT3 is significantly correlated with Gleason score and the levels of upstream regulators (Cdk5 and p35) as well as downstream protein (AR). In conclusion, this study demonstrates that Cdk5 regulates STAT3 activation through S727 phosphorylation and further promotes AR activation by protein-protein interaction in prostate cancer cells.

    August 13, 2013   doi: 10.1152/ajpendo.00615.2012   open full text
  • Slow Oscillations of KATP Conductance in Mouse Pancreatic Islets Provide Support for Electrical Bursting Driven by Metabolic Oscillations.
    Ren, J., Sherman, A., Bertram, R., Goforth, P. B., Nunemaker, C. S., Waters, C. D., Satin, L. S.
    AJP Endocrinology and Metabolism. August 06, 2013

    We used the patch clamp technique in situ to test the hypothesis that slow oscillations in metabolism mediate slow electrical oscillations in mouse pancreatic islets by causing oscillations in KATP channel activity. Total conductance was measured over the course of slow bursting oscillations in surface β-cells of islets exposed to 11.1 mM glucose by either switching from current clamp to voltage clamp at different phases of the bursting cycle or by clamping the cells to –60 mV and running two-second voltage ramps from –120 to –50 mV every 20 seconds. The membrane conductance, calculated from the slopes of the ramp current-voltage curves, oscillated and was larger during the silent phase than during the active phase of the burst. The ramp conductance was sensitive to diazoxide, and the oscillatory component was reduced by sulfonylureas or by lowering extracellular glucose to 2.8 mM, suggesting that the oscillatory total conductance is due to oscillatory KATP channel conductance. We demonstrate that these results are consistent with the Dual Oscillator Model, in which glycolytic oscillations drive slow electrical bursting, but not with other models in which metabolic oscillations are secondary to calcium oscillations. The simulations also confirm that oscillations in membrane conductance can be well estimated from measurements of slope conductance and distinguished from gap junction conductance. The data, combined with insights from mathematical models, support a mechanism of slow (~5 min) bursting driven by oscillations in metabolism, rather than by oscillations in the intracellular free calcium concentration.

    August 06, 2013   doi: 10.1152/ajpendo.00046.2013   open full text
  • Diet reduction in obese ewes from early gestation prevents glucose-insulin dysregulation and returns fetal adiposity and organ development to control levels.
    Tuersunjiang, N., Odhiambo, J. F., Long, N. M., Shasa, D. R., Nathanielsz, P. W., Ford, S. P.
    AJP Endocrinology and Metabolism. August 06, 2013

    Maternal obesity poses significant risks for offspring health. This study evaluated the effects of reducing dietary intake of obese overfed ewes from early gestation on fetal growth and development. Sixty days prior to conception ewes were assigned to a control diet [CON: 100% of National Research Council (NRC) recommendations], a diet inducing maternal obesity [MO: 150% NRC] or a maternal obesity intervention diet [MOI: 150% NRC to day 28 of gestation then 100% NRC thereafter] until necropsy at mid (day 75) or late (day 135) gestation. Fetal size and weight, as well as fetal organ weights were greater (P < 0.05) at mid gestation in MO ewes than those of CON and MOI ewes. By late gestation, while fetal size and weight did not differ among dietary groups, cardiac ventricular weights and wall thicknesses, as well perirenal fat weights remained elevated in fetuses from MO ewes compared to those from CON and MOI ewes. At both mid- and late gestation, MO fetuses exhibited elevated (P < 0.05) plasma concentrations of triglycerides, cholesterol, insulin, and cortisol compared to CON and MOI fetuses. While plasma glucose was reduced in CON and MOI versus MO fetuses in mid-gestation, glucose concentrations in MOI fetuses increased to those of MO fetuses by late gestation in association with increased placental GLUT3 expression. These data are consistent with the concept that reducing maternal diet of obese overfed ewes to requirements from early gestation can prevent MO induced alterations in fetal growth and adiposity through late gestation.

    August 06, 2013   doi: 10.1152/ajpendo.00117.2013   open full text
  • Impact of embryo number and periconceptional undernutrition on factors regulating adipogenesis, lipogenesis and metabolism in adipose tissue in the sheep fetus.
    Lie, S., Morrison, J. L., Williams-Wyss, O., Ozanne, S. E., Zhang, S., Walker, S. K., Kleeman, D. O., MacLaughlin, S. M., Roberts, C. T., McMillen, I. C.
    AJP Endocrinology and Metabolism. August 06, 2013

    Maternal undernutrition around the time of conception is associated with increased risk of insulin resistance in adulthood. We hypothesised that maternal undernutrition during the periconceptional (PCUN: -60-7d) and/or preimplantation (PIUN: 0-7d) periods would result in a decrease in UCP1 expression and the abundance of insulin signalling molecules and increased abundance of factors which regulate adipogenesis and lipogenesis in fetal perirenal adipose tissue (PAT) and that these effects would be different in singletons and twins. Maternal PCUN and PIUN resulted in a decrease in UCP1 expression in PAT and PIUN resulted in higher circulating insulin concentrations, an increased abundance of pPKC and PDK4 and a decreased abundance of Akt1, phosphorylated mTOR and PPAR in PAT in singleton and twin fetuses. In singletons there was also a decrease in the abundance of p110β in PAT in the PCUN and PIUN groups and an increase in total AMPKα in PAT in the PIUN group. In twins, however, there was an increase in the abundance of mTOR in the PCUN group and an increase in PDK2 and decrease in total AMPK α in the PIUN group. Thus exposure to periconceptional undernutrition programs changes in the thermogenic capacity and the insulin and fatty acid oxidation signalling pathway in visceral fat, and these effects are different in singletons and twins. These findings are important as the thermogenic capacity of brown fat and the insulin sensitivity of visceral fat are important determinants of the risk of developing obesity and an insulin resistance phenotype in later life.

    August 06, 2013   doi: 10.1152/ajpendo.00180.2013   open full text
  • The permissive role of prolactin as a regulator of luteinizing hormone action in the female mouse ovary and extragonadal tumorigenesis.
    Bachelot, A., Carre, N., Mialon, O., Matelot, M., Servel, N., Monget, P., Ahtiainen, P., Huhtaniemi, I. T., Binart, N.
    AJP Endocrinology and Metabolism. August 06, 2013

    Transgenic female mice overexpressing the hCGβ subunit (hCGß+) and producing elevated levels of LH/hCG bioactivity, present as young adults with enhanced ovarian steroidogenesis, precocious puberty and infertility. They subsequently develop pituitary prolactinomas, high circulating prolactin (PRL) levels and marked mammary gland lobuloalveolar development followed by adenocarcinomas. None of these phenotypes appear in gonadectomized mice, indicating that the hCG-induced aberrations of ovarian function are responsible for the extragonadal phenotypes. PRL receptor deficient (PRLR-/-) female mice are sterile, despite ovulating, due to a failure of embryo implantation, as a consequence of decreased ovarian LH receptor (Lhcgr) expression, and inadequate corpus luteum formation and progesterone production. To study further the presumed permissive role of PRL in the maintenance of gonadal responsiveness to LH/hCG stimulation, we crossed the hCGß+ and PRLR-/- mice. The double mutant hCGß+/PRLR-/- females remained sterile with an ovarian phenotype similar to PRLR-/- mice, indicating that LH action, Lhcgr expression and consequent luteinization are not possible without simultaneous PRL signalling. The high frequency of pituitary prolactinomas in PRL-/- mice was not affected by transgenic hCGβ expression. In contrast, none of the hCGß+/PRLR-/- females showed either mammary gland lobuloalveolar development or tumors, and the increased mammary gland Wnt-5b expression, possibly responsible for the tumorigenesis in hCGβ + mice, was absent in double mutant mice. Hence, high LH/hCG stimulation is unable to compensate for missing PRL signalling in the maintenance of luteal function. PRL thus appears to be a major permissive regulator of LH action in the ovary and of its secondary extragonadal effects.

    August 06, 2013   doi: 10.1152/ajpendo.00243.2013   open full text
  • Acid Sphingomyelinase Plays a Key Role in Palmitic Acid-Amplified Inflammatory Signaling Triggered by Lipopolysaccharide at Low Concentration in Macrophages.
    Jin, J., Zhang, X., Lu, Z., Perry, D. M., Li, Y., Hannun, Y. A., Huang, Y.
    AJP Endocrinology and Metabolism. August 06, 2013

    Periodontal disease is more prevalent and severe in patients with diabetes than that in nondiabetic patients. In addition to diabetes, a large number of studies have demonstrated an association between obesity and chronic periodontal disease. However, the underlying mechanisms have not been well understood. Since plasma free fatty acids (FAs) are elevated in obese patients and saturated FAs such as palmitic acid (PA) have been shown to increase host inflammatory response, we sought to find out how PA interacts with lipopolysaccharide (LPS), an important pathological factor involved in periodontal disease, to enhance inflammation. We found that while low concentration of LPS (1 ng/ml) stimulated interleukin (IL)-6 expression in RAW264.7 macrophages, PA further augmented it by 4 folds. Besides IL-6, PA amplified the stimulatory effect of LPS on a large number of toll-like receptor (TLR)4-mediated expression of pro-inflammatory signaling molecules such as interleukin-1 receptor-associated kinase-like 2 and pro-inflammatory molecules including monocyte chemotactic protein-1 and colony stimulating factor. We also observed that PA augmented TLR4, but not TLR2 signal, and the augmentation was mediated by nuclear factor kappa B (NFB) pathways. To further elucidate the regulatory mechanism whereby PA amplifies LPS signal, our studies showed that PA and LPS synergistically increased hydrolysis of sphingomyelin by stimulating acid sphingomyelinase (ASMase) activity, which contributed to a marked increase in ceramide production and IL-6 upregulation. Taken together, this study has demonstrated that PA markedly augments TLR4-mediated proinflammatory signaling triggered by low concentration of LPS in macrophages and ASMase plays a key role in the augmentation.

    August 06, 2013   doi: 10.1152/ajpendo.00251.2013   open full text
  • APPL1 transgenic mice are protected from high fat diet induced cardiac dysfunction.
    Park, M., Wu, D., Park, T., Choi, C.-s., Li, R., Cheng, K. K. Y., Xu, A., Sweeney, G.
    AJP Endocrinology and Metabolism. August 06, 2013

    APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper motif-1) has been established as an important mediator of insulin and adiponectin signaling. Here we investigated the influence of transgenic (Tg) APPL1 overexpression in mice on high fat diet (HFD) induced cardiomyopathy in mice. Wild type (Wt) mice fed a high fat diet for 16 weeks showed cardiac dysfunction, determined by echocardiography, with decreased ejection fraction, decreased fractional shortening and increased end diastolic volume. HFD-fed APPL1 Tg mice were significantly protected from this dysfunction. Speckle tracking echocardiography to accurately assess cardiac tissue deformation strain and wall motion also indicated dysfunction in Wt mice and a similar improvement in Tg versus Wt mice on HFD. APPL1 Tg mice had less HFD-induced increase in circulating non-esteridied fatty acid levels and myocardial lipid accumulation. Lipidomic analysis using LC-MS/MS showed HFD significantly increased myocardial contents of distinct ceramide, sphingomyelin and diacylglycerol (DAG) species, of which increases in C16:0 and C18:0 ceramides plus C16:0 and C18:1 DAGs were attenuated in Tg mice. Glucose tolerance test indicated less peripheral insulin resistance in response to HFD in Tg mice which was also apparent by measuring cardiac Akt phosphorylation and cardiomyocyte glucose uptake. In summary, APPL1 Tg mice exhibit improved peripheral metabolism, reduced cardiac lipotoxicity and improved insulin sensitivity. These cellular effects contribute to protection from HFD-induced cardiomyopathy.

    August 06, 2013   doi: 10.1152/ajpendo.00257.2013   open full text
  • Whole body metabolic effects of prolonged endurance training in combination with erythropoietin treatment in humans: a randomized placebo controlled study.
    Christensen, B., Nellemann, B., Larsen, M. S., Thams, L., Sieljacks, P., Vestergaard, P. F., Bibby, B. M., Vissing, K., Stodkilde-Jorgensen, H., Pedersen, S. B., Moller, N., Nielsen, S., Jessen, N., Jorgensen, J. O. L.
    AJP Endocrinology and Metabolism. August 06, 2013

    Erythropoietin (Epo) administration improves aerobic exercise capacity and insulin sensitivity in renal patients, and also increases resting energy expenditure (REE). Similar effects are observed in response to endurance training. The aim was to compare the effects of endurance training with erythropoiesis-stimulating agent (ESA) treatment in healthy humans. Thirty-six healthy untrained men were randomized to 10 weeks of either: 1) placebo (n=9), 2) ESA (n=9), 3) endurance training (n=10), or 4) ESA and endurance training (n=8). In a single blinded design, ESA/placebo was injected once weekly. Training consisted of biking for 1h at 65% of wattmax 3 times/week. Measurements performed before and after the intervention: body composition, maximal oxygen uptake, insulin sensitivity, REE, and palmitate turnover. Uncoupling protein 2 (UCP2) mRNA levels were assessed in skeletal muscle. Fat mass decreased after training (p=0.003), whereas ESA induced a small but significant increase in intrahepatic fat (p=0.025). Serum free fatty-acid (FFA) levels and palmitate turnover decreased significantly in response to training, whereas the opposite pattern was found after ESA. REE corrected for lean body mass increased in response to ESA and training and muscle UCP2 mRNA levels increased after ESA (p=0.035). Insulin sensitivity increased only after training (p=0.011). Conclusions: 1) Insulin sensitivity is not improved after ESA treatment despite improved exercise capacity, 2) The calorigenic effects of ESA may be related to increased UCP2 gene expression in skeletal muscle, 3) Training and ESA exert opposite effects on lipolysis under basal conditions, increased FFA levels and liver fat fraction was observed after ESA treatment.

    August 06, 2013   doi: 10.1152/ajpendo.00269.2013   open full text
  • Materno-Fetal Transfer of Docosahexaenoic Acid (DHA) is Impaired by Gestational Diabetes Mellitus.
    Pagan, A., Prieto-Sanchez, M. T., Blanco-Carnero, J. E., Gil-Sanchez, A., Parrilla, J. J., Demmelmair, H., Koletzko, B., Larque, E.
    AJP Endocrinology and Metabolism. August 06, 2013

    Better knowledge on the disturbed mechanisms implicated in materno-fetal long-chain polyunsaturated fatty acid (LC-PUFA) transfer in pregnancies with gestational diabetes mellitus (GDM) may have potential high implications for later on in effective LC-PUFA supplementation. We studied in vivo placental transfer of fatty acids (FA) using stable isotopes tracers, administrated to 11 control and 9 GDM pregnant women (6 treated with insulin). Subjects received orally 13C-palmitic, 13C-oleic and 13C-linoleic acids and 13C-docosahexaenoic acid (13C-DHA) 12h before elective caesarean section. Maternal blood samples were collected at -12,-3,-2,-1, delivery and +1h. Placental tissue and venous cord blood were also collected. FA were quantified by gas chromatography (GC) and 13C-enrichments by GC-isotope ratio mass spectrometry. 13C-FA concentration was higher in total lipids of maternal plasma in GDM vs. controls, except for 13C-DHA. Moreover, 13C-DHA showed lower placenta/maternal plasma ratio in GDM vs. controls and significantly lower cord/maternal plasma ratio. For the other studied FA ratios were not different between GDM and controls. Disturbed 13C-DHA placental uptake occurs in both GDM treated with diet or insulin, while the last ones also have lower 13C-DHA in venous cord. The tracer study pointed towards impaired placental DHA uptake as critical step, while the transfer of the rest of 13C-FA was less affected. GDM under insulin treatment could have also higher fetal fat storage contributing to reduce 13C-DHA in venous cord. DHA transfer to the fetus was reduced in GDM pregnancies compared to controls which might affect the programming of neurodevelopment in their neonates.

    August 06, 2013   doi: 10.1152/ajpendo.00291.2013   open full text
  • Long-term exposure to a high-fat diet results in the development of glucose intolerance and insulin resistance in interleukin-1 receptor I deficient mice.
    McGillicuddy, F. C., Reynolds, C. M., Finucane, O., Coleman, E., Harford, K. A., Grant, C., Sergi, D., Williams, L. M., Mills, K. H., Roche, H. M.
    AJP Endocrinology and Metabolism. August 06, 2013

    Aims: Emerging evidence has demonstrated that saturated fatty acids (SFA) prime pro-IL-1β production and inflammasome-mediated IL-1β activation is critical in obesity-associated insulin resistance (IR). Nonetheless, IL-1 receptor I deficient (IL-1RI-/-) mice develop mature-onset obesity despite consuming a low-fat diet (LFD). With this apparent contradiction, the present study evaluated whether IL-1RI-/- mice were protected against long-term (6 months (6mo)) HFD-induced IR. Methods: Male wild-Type (WT) and IL-1RI-/- mice were fed LFD or HFD for 3 months (3mo) or 6mo and glucose (GTT) and insulin (ITT) tolerance tests performed. Adipose insulin sensitivity, cytokine profiles and adipocyte morphology were assessed. The adipogenic potential of stromal vascular fraction (SVF) was determined. Hepatic lipid accumulation and insulin sensitivity were characterized. Results: IL-1RI-/- mice developed glucose intolerance and IR after 6mo HFD compared to 3mo HFD, coincident with enhanced weight-gain, hyperinsulinemia and hyperleptinemia. The aggravated IR phenotype was associated with loss of adipose functionality, switch from adipocyte hyperplasia to hypertrophy and hepatosteatosis. Induction of adipogenic genes was reduced in IL-1RI-/- pre-adipocytes after 6mo HFD compared to 3mo HFD. Obese LFD-IL-1RI-/- mice exhibited preserved metabolic health. Conclusions: IL-1RI-/- mice develop glucose intolerance and IR after 6mo HFD intervention. While mature-onset obesity is evident in LFD-IL-1RI-/- mice, the additional metabolic insult of HFD was required to drive adipose inflammation and systemic IR. These findings indicate an important interaction between dietary fat and IL-1, relevant to optimal metabolic health.

    August 06, 2013   doi: 10.1152/ajpendo.00297.2013   open full text
  • Androgens influence microvascular dilation in PCOS through ET-A and ET-B receptors.
    Wenner, M. M., Taylor, H. S., Stachenfeld, N. S.
    AJP Endocrinology and Metabolism. August 06, 2013

    Hyperandrogenism and vascular dysfunction often coexist in women with Polycystic Ovary Syndrome (PCOS). We hypothesized that testosterone compromises cutaneous microvascular dilation in women with PCOS via the endothelin-1 ET-B subtype receptor. To control and isolate testosterone effects on microvascular dilation, we administered a gonadotropin releasing hormone antagonist (GnRHant) for 11 days in obese otherwise healthy women [(Controls, 22.0 (4) y, 36.0 (3.2) kg/m2)] or women with PCOS [23 (4) y, 35.4 (1.3) kg/m2)], adding testosterone (T, 2.5 mg/day) for days 8-11. Using laser Doppler flowmetry and cutaneous microdialysis, we measured changes in skin microcirculatory responsiveness (CVC) to local heating while perfusing ET-A (BQ-123) and ET-B (BQ-788) receptor antagonists under three experimental conditions: baseline (BL, pre-hormone intervention), GnRHant (day 4 of administration), and T administration. At BL, ET-A receptor inhibition enhanced heat-induced vasodilation in both groups [CVC Control 2.03 (0.65), PCOS 2.10 (0.25), AU/mmHg, P< 0.05]; ET-B receptor inhibition reduced vasodilation in Controls only [CVC 0.98 (0.39), 1.41 (0.45) AU/mmHg for Controls, PCOS] compared to saline [CVC Controls 1.27 (0.48), PCOS 1.31 (0.13) AU/mmHg]. GnRHant enhanced vasodilation in PCOS [saline CVC 1.69 (0.23) AU/mmHg vs. BL, P<0.05), and abolished the ET-A effect in both groups; a response reasserted with T in Controls. ET-B receptor inhibition reduced heat-induced vasodilation in both groups during GnRHant and T [CVC, Controls: 0.95 (0.21) vs. 0.51 (13), PCOS: 1.27 (0.23) vs. 0.84 (0.27); for GnRHant vs. T, P< 0.05)]. These data demonstrate androgen suppression improves microvascular dilation in PCOS via ET-A and ET-B receptors.

    August 06, 2013   doi: 10.1152/ajpendo.00343.2013   open full text
  • Protein kinase C-induced Activin A Switches Adrenocortical Steroidogenesis to Aldosterone by Suppressing CYP17A1 expression.
    Hofland, J., Steenbergen, J., Hofland, L. J., M. van Koetsveld, P., Eijken, M., van Nederveen, F. H., Kazemier, G., de Herder, W. W., Feelders, R., de Jong, F. H.
    AJP Endocrinology and Metabolism. July 30, 2013

    Functional zonation of the adrenal cortex is a consequence of the zone-specific expression of P450c17 (CYP17A1) and its cofactors. Activin and inhibin peptides are differentially produced within the zones of the adrenal cortex and have been implicated in steroidogenic control. In this study we investigated whether activin and inhibin can function as intermediates in functional zonation of the human adrenal cortex. Activin A suppressed CYP17A1 expression and P450c17 function in adrenocortical cell lines as well as in primary adrenal cell cultures. Inhibin βA-subunit mRNA and activin A protein levels were found to be increased up to 1900-fold and 49-fold, respectively, after protein kinase C (PKC) stimulation through PMA or angiotensin II in H295R adrenocortical carcinoma cells. This was confirmed in HAC15 cells and for PMA in primary adrenal cell cultures. PMA and AngII both decreased CYP17A1 expression in the adrenocortical cell lines, whereas PMA concurrently suppressed CYP17A1 levels in the primary cultures. Inhibition of activin-signaling during PKC stimulation through silencing of the inhibin βA-subunit or blocking of the activin type I receptor opposed the PMA-induced downregulation of CYP17A1 expression and P450c17 function. In contrast, PKA stimulation through adrenocorticotrophin or forskolin increased expression of the inhibin α-subunit and betaglycan, both antagonists of activin action. These data indicate that activin A acts as a PKC-induced paracrine factor involved in the suppression of CYP17A1 in the zona glomerulosa and thereby can contribute to functional adrenocortical zonation.

    July 30, 2013   doi: 10.1152/ajpendo.00034.2013   open full text
  • GCN2 regulates the CCAAT enhancer binding protein beta and hepatic gluconeogenesis.
    Xu, X., Hu, J., McGrath, B. C., Cavener, D. R.
    AJP Endocrinology and Metabolism. July 30, 2013

    Mice deficient for Gcn2 either globally or specifically in the liver display reduced capacity to maintain glucose homeostasis during fasting suggesting the hypothesis that GCN2 may regulate gluconeogenesis (GNG), which normally plays a key role maintaining peripheral glucose homeostasis. Gcn2 deficient mice exhibit normal insulin sensitivity and plasma insulin, but show reduced gluconeogenesis when administered pyruvate, a gluconeogenic substrate. The basal expression of PEPCK, a rate-limiting enzyme in gluconeogenesis, is abnormally elevated in Gcn2 KO mice in the fed state but fails to be further induced during fasting. The level of TCA cycle intermediates, including malate and oxaloacetate, and the NADH/NAD+ ratio are perturbed in the liver of Gcn2 KO mice either in the fed or fasted state, which may directly impinge upon gluconeogenesis. Additionally, the expression of the CCAAT enhancer binding protein beta (C/EBPβ) in the liver fails to be induced in Gcn2 KO mice after 24hr fasting and the liver-specific Cebpβ KO mice show reduced fasting GNG similar to that seen in Gcn2 deficient mice. Our study demonstrates that GCN2 is important in maintaining GNG in the liver, which is likely to be mediated through regulation of C/EBPβ.

    July 30, 2013   doi: 10.1152/ajpendo.00063.2013   open full text
  • Dietary protein decreases exercise endurance through rapamycin-sensitive suppression of muscle mitochondria.
    Mitsuishi, M., Miyashita, K., Muraki, A., Tamaki, M., Tanaka, K., Itoh, H.
    AJP Endocrinology and Metabolism. July 30, 2013

    Loss of physical performance is linked not only to decreased activity in daily life but also to increased onset of cardiovascular diseases and mortality. A high-protein diet is recommended for aged individuals in order to preserve muscle mass; however, the regulation of muscle mitochondria by dietary protein has not been clarified. We investigated the long-term effects of a high-protein diet on muscle properties, focusing especially on muscle mitochondria. Mice were fed a high-protein diet from the age of 8 weeks and examined for mitochondrial properties and exercise endurance at the ages of 20 and 50 weeks. Compared to normal chow, a high-protein diet significantly decreased the amount of muscle mitochondria, the mitochondrial activity and the running distance at 50 weeks, although it increased muscle mass and grip power. Inhibition of TORC1-dependent signal pathways by rapamycin from 8 weeks suppressed the decline in mitochondria and exercise endurance observed when mice were fed the high-protein diet in association with preserved AMPK activity. Collectively, these findings suggest a role for dietary protein as a suppressor of muscle mitochondria and indicate that the age-associated decline in exercise endurance might be accelerated by excessive dietary protein through rapamycin-sensitive suppression of muscle mitochondria.

    July 30, 2013   doi: 10.1152/ajpendo.00145.2013   open full text
  • Adult-onset obesity induced by early life overnutrition could be reversed by moderate caloric restriction.
    Liu, H.-W., Srinivasan, M., Mahmood, S., Smiraglia, D. J., Patel, M. S.
    AJP Endocrinology and Metabolism. July 30, 2013

    Overnutrition during the suckling period (small litter, SL) results in the development of adult-onset obesity. Our aim was to investigate if two levels of caloric restriction (CR) in the early postweaning period can reverse obese phenotype in SL rats. The normal litter (NL) had 12 pups/dam and SL had 3 male pups/dam from the postnatal day 3 until 21. After weaning rats consumed lab-chow as indicated: (i) NL and SL groups were on ad libitum regimen up to day 140, (ii) another SL group was pair-fed (SL/PF) to NL(~14% reduction), (iii) SL/PF/AL group was pair-fed up to day 94 and then switched to ad libitum feeding, (iv) SL/CR group received 24% reduction (moderate CR) in food intake compared to SL, and (v) SL/CR/AL group was on 24% CR up to day 94 and then switched to ad libitum feeding. Pair-feeding reduced body weight gains and serum insulin and leptin levels compared to SL rats, but these parameters were restored to SL levels in the SL/PF/AL rats after switching to ad libitum feeding. Interestingly, the moderate CR normalized these parameters in SL/CR and SL/CR/AL rats compared to NL. The expression of neuropeptide Y, pro-opiomelanocortin and leptin receptor returned to control levels in the hypothalami from SL/CR and SL/CR/AL rats. These results indicate that appropriate manipulation of energy intake during the early postweaning period could lead to longer lasting effects on the regulation of body weight homeostasis via reversal of the early preweaning programming effects on the hypothalamic appetite regulation mechanism.

    July 30, 2013   doi: 10.1152/ajpendo.00280.2013   open full text
  • Local NOS inhibition impairs vascular and metabolic actions of insulin in rat muscle in vivo.
    Bradley, E. A., Richards, S. M., Keske, M. A., Rattigan, S.
    AJP Endocrinology and Metabolism. July 30, 2013

    Insulin stimulates microvascular recruitment in skeletal muscle and this vascular action augments muscle glucose disposal by ~40%. The aim of the current study was to determine the contribution of local nitric oxide synthase (NOS) to the vascular actions of insulin in muscle. Hooded Wistar rats were infused with the NOS inhibitor N-L-nitro-arginine-methyl ester (L-NAME, 10µM) retrogradely via the epigastric artery into one leg during a systemic hyperinsulinemic euglycaemic clamp ( 60 min) or saline infusion. Femoral artery blood flow, microvascular blood flow (assessed from 1-methyl xanthine metabolism) and muscle glucose uptake (2-deoxyglucose uptake) was measured in both legs. Local L-NAME infusion did not have any systemic actions on blood pressure or heart rate. Local L-NAME blocked insulin-stimulated changes in femoral artery blood flow (84%, P<0.05) and microvascular recruitment (98%, P<0.05), and partially blocked insulin-mediated glucose uptake in muscle (reduced by 34%, P< 0.05). L-NAME alone did not have any metabolic effects in the hindleg. We conclude that insulin-mediated microvascular recruitment is dependent on local activation of NOS in muscle, and that this action is important for insulin's metabolic actions.

    July 30, 2013   doi: 10.1152/ajpendo.00289.2013   open full text
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